Iron exacerbates congenital cholestatic liver injury via bile acid-induced ferroptosis

Iron exacerbates congenital cholestatic liver injury via bile acid-induced ferroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Iron exacerbates congenital cholestatic liver injury via bile acid-induced ferroptosis Toshiro Moroishi, Yudai OHTA, Yohei KANAMORI, Ayato MAEDA, Mohamed Fathi Saleh, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6671814/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pediatric cholestatic liver diseases are rare but serious conditions that frequently progress to liver fibrosis and cirrhosis and often require transplantation. Despite their clinical importance, the mechanisms driving disease progression remain poorly understood. Here, we report that hepatic iron accumulation is a pathological feature associated with congenital cholestatic liver disease in mice with a liver-specific deletion of Yap , a gene critical for bile duct development. We demonstrated that further hepatic iron overload induced by liver-specific deletion of Fbxl5 , a key regulator of cellular iron homeostasis, exacerbated cholestatic liver injury and fibrosis in Yap -deficient mice. Mechanistically, iron overload enhanced the susceptibility to bile acid-induced cytotoxicity via ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation. This ferroptotic process was confirmed by the suppression of bile acid-induced cell death through iron chelation and lipid peroxide scavenging in ex vivo liver slice cultures. Furthermore, both dietary iron restriction and antioxidant treatment mitigated liver injury in vivo . These findings identify iron accumulation as a key driver of disease progression and highlight iron metabolism and ferroptosis as potential therapeutic targets in congenital cholestatic liver disease. Biological sciences/Cell biology/Cell death Biological sciences/Biochemistry/Metals/Iron Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Cholestatic liver diseases in the neonatal period typically present with cholestasis as an initial symptom, accompanied by inflammation and liver fibrosis, which ultimately progress to liver cirrhosis in severe cases. Congenital disorders associated with bile duct malformations, such as biliary atresia and Alagille syndrome, represent the most common causes of pediatric cholestasis 1 . Approximately half of modern pediatric liver transplantations are related to cholestatic liver diseases 2 , and medical treatments for these conditions remain insufficiently established. Intrahepatic bile ducts in mice originate from hepatoblasts, the common progenitors of the hepatocyte and cholangiocyte lineages, and differentiate between embryonic day 11.5 and 13.5. Several studies have indicated that Yes-associated protein (YAP), a transcriptional coactivator in the Hippo intracellular signaling pathway 3 , regulates this differentiation process 4 , 5 . YAP is widely recognized as a critical determinant of cell fate, modulating both cell differentiation and proliferation in diverse contexts, including cancer progression and embryonic development 6 , 7 . During liver development, YAP has been suggested to promote the differentiation of hepatoblasts into cholangiocytes through interactions with SRY-box transcription factor 9 (SOX9) and the Notch signaling pathway 5 , 8 . Indeed, the loss of YAP during liver development leads to defects of biliary ductal structures and a marked paucity of bile ducts, causing long-term cholestatic injury 4 , 8 . Although the underlying causes and pathological mechanisms of cholestasis in pediatric cholestatic liver disease are diverse, obstruction of bile flow and subsequent accumulation of intrahepatic bile components are common features. However, the detailed mechanisms underlying the progression of pediatric cholestatic liver disease remain unclear. Iron overload is a risk factor for worsening disease outcomes in various chronic liver diseases, such as hepatitis C virus infection (HCV) 9 , alcoholic liver disease 10 , and metabolic dysfunction-associated steatohepatitis (MASH) 11 . Iron overload under pathological conditions leads to an imbalance in iron homeostasis and causes excessive ferrous iron (Fe 2+ ) accumulation. Fe 2+ facilitates reactive oxygen species (ROS) production through the Fenton reaction, resulting in lipid peroxidation and ultimately triggering ferroptosis, a recently identified form of regulated cell death distinct from apoptosis and necrosis, characterized by iron-dependent lipid peroxide accumulation 3 . This process has been suggested to be a critical mechanism by which iron overload contributes to the progression of liver diseases 11 – 15 . Despite its relevance in other liver disease, the involvement of ferroptosis in the pathogenesis of congenital cholestatic liver disease remains poorly understood. Cellular iron homeostasis is primarily regulated by the iron-sensing protein F-box and leucine-rich protein 5 (FBXL5) and the RNA-binding protein iron regulatory protein 2 (IRP2) 16 , 17 . IRP2 post-transcriptionally upregulates the expression of proteins responsible for iron uptake and simultaneously suppresses the expression of proteins involved in iron export, thereby increasing the cellular labile iron pool (Fe 2+ ). FBXL5 is a substrate-recognition subunit of the SCF-type E3 ubiquitin ligase complex, facilitating the recruitment of IRP2 for its ubiquitination and subsequent degradation. FBXL5 binds directly to iron and is stabilized when intracellular iron is abundant, leading to the degradation of IRP2, which helps reduce cellular iron levels and maintain iron homeostasis 16 , 17 . FBXL5 deficiency stabilizes IRP2 regardless of cellular iron levels, causing cellular iron overload. FBXL5-null mice display early embryonic lethality resulting from iron-induced oxidative stress, which is rescued by the additional deletion of IRP2, suggesting that the precise regulation of IRP2 by FBXL5 is crucial for maintaining cellular iron homeostasis 18 . Additionally, studies using mice with a conditional deletion of FBXL5 in different cell types have demonstrated that FBXL5 deficiency leads to increased cellular iron levels, which is attributed to the aberrant accumulation of IRP2 in vivo 18 – 21 . In this study, we showed that hepatic iron accumulation correlates with cholestasis progression in mice with biliary malformations induced by hepatic YAP deficiency. Hepatic iron overload caused by FBXL5 depletion augmented congenital cholestatic liver disease phenotypes in hepatic YAP-deficient mice. Mechanistically, bile acid toxicity was exacerbated by ferroptosis, driven by iron accumulation, which accelerated liver injury and contributed to disease progression. This study provides insights into the role of iron as a pathogenic factor in congenital chronic cholestatic liver diseases. RESULTS Hepatic iron accumulation is a hallmark of cholestatic liver disease induced by YAP deficiency To investigate the pathogenesis of congenital cholestatic liver disease, we generated mice with liver-specific ablation of Yap by crossing Yap flox/flox mice with Albumin -Cre ( Alb -Cre) transgenic mice. Loss of YAP protein in the liver tissue was confirmed by immunoblotting (Supplementary Fig. 1A). The injection of India ink into the extrahepatic bile ducts of control ( Yap flox/flox ) mice resulted in the flow of ink into intrahepatic bile ducts, allowing visualization of the interlobular bile ducts and bile canaliculi. In contrast, consistent with previous observations 4 , 5 , Alb -Cre/ Yap flox/flox [hereafter referred to as YAP liver-KO (knockout)] mice showed no ink flow into the intrahepatic bile ducts (Supplementary Fig. 1B). Furthermore, immunostaining of biliary epithelial cells revealed well-organized tubular structures in control livers, whereas YAP-deficient livers exhibited obstructed duct-like structures (Supplementary Fig. 1C). Histological analysis revealed multiple areas of hepatic cell death in YAP-deficient livers (Supplementary Fig. 1C). These pathological features, including impaired bile flow, bile duct obstruction, and hepatocellular injury, demonstrated that YAP deletion during development recapitulates the key characteristics of congenital cholestatic liver disease. To explore potential pathogenic factors contributing to disease progression, we assessed hepatic iron levels in YAP liver-KO mice. Measurement of non-heme iron content using a bathophenanthroline-based colorimetric assay revealed significantly elevated hepatic iron levels in YAP liver-KO mice compared to those in control mice (Fig. 1 A). Diaminobenzidine (DAB)-enhanced Perls' staining further confirmed that iron accumulation was predominantly localized to the hepatocytes of YAP liver-KO mice (Fig. 1 B). Notably, the degree of hepatic iron accumulation correlated with serum bilirubin levels in YAP liver-KO mice (Fig. 1 C), suggesting that iron deposition is associated with the progression of cholestatic liver disease in this model. Genetically induced iron overload exacerbates cholestatic liver disease progression in YAP-deficient mice To investigate the role of iron in the pathologies of congenital cholestatic liver disease, we induced hepatic iron overload in YAP liver-KO mice by crossing them with Fbxl5 flox/flox mice. Consistent with previous findings 18 , FBXL5 deficiency led to the hepatic accumulation of IRP2 (Supplementary Fig. 2A) and subsequent iron overload (Fig. 2 A) in both FBXL5 liver-knockout ( Alb -Cre /Fbxl5 flox/flox ) and YAP/FBXL5 double knockout ( Alb -Cre/ Yap flox/flox / Fbxl5 flox/flox ) mice, hereafter referred to as FBXL5 liver-KO and YAP/FBXL5 liver-dKO mice, respectively. FBXL5 liver-KO mice displayed no signs of cholestatic liver disease, including jaundice, hyperbilirubinemia, or liver fibrosis. In contrast, YAP/FBXL5 liver-dKO mice exhibited more pronounced jaundice than YAP liver-KO mice at 10 weeks of age (Fig. 2 B). Serum analyses confirmed that cholestasis was significantly exacerbated in YAP/FBXL5 liver-dKO mice, as indicated by elevated total and conjugated bilirubin levels compared to those in YAP liver-KO mice (Fig. 2 C). Furthermore, Sirius red staining revealed enhanced liver fibrosis in YAP/FBXL5 liver-dKO mice compared to that in YAP liver-KO mice (Fig. 2 D). These results indicate that iron accumulation resulting from FBXL5 deficiency aggravates cholestatic liver injury and fibrosis in the context of YAP deficiency. Iron overload enhances liver injury in YAP-deficient mice To elucidate how iron accumulation contributes to the progression of congenital cholestatic liver disease, we analyzed temporal changes in the severity of cholestasis. From 2 to 8 weeks of age, total bilirubin levels were persistently elevated in both YAP liver-KO and YAP/FBXL5 liver-dKO mice compared to those in control and FBXL5 liver-KO mice (Fig. 3 A). Notably, while bilirubin levels were comparable between YAP liver-KO and YAP/FBXL5 liver-dKO mice at 2 weeks, significantly higher levels were observed in dKO mice at 4 and 8 weeks. These observations suggested that pathological events occurring as early as 2 weeks of age may underlie iron overload-driven exacerbation of cholestatic liver disease in YAP-deficient livers. Given that liver injury caused by cholestasis can disrupt the intrahepatic bile ducts and accelerate disease progression 22 , we assessed liver damage at 2 weeks of age. While serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were not significantly different among control, FBXL5 liver-KO, and YAP liver-KO mice, YAP/FBXL5 liver-dKO mice exhibited significantly elevated levels of both ALT and ALP (Fig. 3 B). Histological analysis confirmed extensive hepatocyte death in the livers of YAP/FBXL5 liver-dKO mice (Fig. 3 C). These results suggested that hepatic iron overload caused by FBXL5 deficiency aggravates early liver injury, thereby accelerating the progression of cholestatic liver disease. Iron increases susceptibility to bile acid toxicity To investigate the molecular mechanism by which iron overload exacerbates liver injury in YAP-deficient livers, we examined the interplay between iron and bile acid toxicity. Previous studies have demonstrated that the cytotoxic effects of bile acids primarily drive hepatocellular and biliary injuries in cholestatic liver disease 23 . To assess how iron influences bile acid-induced cell death, we utilized ex vivo precision-cut liver slices (PCLS), a 3D culture system that preserves the native liver architecture, and stimulated them with deoxycholic acid (DCA) 23 – 25 , a representative hydrophobic and toxic bile acid implicated in cholestatic liver injury. We first established a dose-response relationship using PCLS in control mice. Cell death, assessed using propidium iodide (PI) staining, was induced by DCA at concentrations ≥ 1 mM, while concentrations < 800 µM had minimal cytotoxic effects (Supplemental Figure. 3A). Based on this, we selected 750 µM DCA—a sublethal dose for control tissue—to assess the impact of iron overload on bile acid sensitivity. We then examined the response to PCLS in 2-week-old FBXL5 liver-KO, YAP liver-KO, and YAP/FBXL5 liver-dKO mice. Exposure to 750 µM DCA caused only mild cell death (~ 20%) in YAP liver-KO slices, but resulted in marked cell death (> 60%) in PCLS from both FBXL5 liver-KO and YAP/FBXL5 liver-dKO mice (Fig. 4 B). Notably, the extent of DCA-induced cell death was similar between FBXL5 liver-KO and YAP/FBXL5 liver-dKO PCLS, indicating that iron overload due to FBXL5 deficiency, rather than YAP loss, is the primary factor sensitizing the liver tissue to bile acid toxicity. Furthermore, treatment of control PCLS with ferric ammonium citrate (FAC), an exogenous iron source, similarly enhanced DCA-induced cell death (Fig. 4 C). Collectively, these results suggested that hepatic iron overload enhances the cytotoxic effects of bile acids and contributes to the progression of liver injury in cholestatic liver disease. Toxic bile acid induces ferroptosis in iron-loaded cells Given that excess iron is a known driver of ferroptosis 3 , we hypothesized that liver tissue with iron overload would exhibit increased susceptibility to ferroptosis. To test this, we evaluated whether bile acid-induced cell death involves ferroptosis in the context of iron overload. PCLS from FBXL5 liver-KO mice were exposed to DCA in the presence or absence of two established ferroptosis inhibitors 3 : the iron chelator deferoxamine (DFO) and the lipid peroxidation scavenger ferrostatin-1 (Fer-1). Consistent with previous observations, DCA exposure induced substantial cell death (> 60%) in PCLS from FBXL5-deficient mice. However, treatment with DFO (Fig. 5 A) or Fer-1 (Fig. 5 B) significantly reduced DCA-induced cell death, indicating that cytotoxicity depended on both iron and lipid peroxidation, which are hallmarks of ferroptosis. Supporting this interpretation, immunohistochemical analysis revealed marked accumulation of 4-hydroxynonenal (4-HNE), a well-established lipid peroxidation product and ferroptosis marker 26 , in liver tissues from 2-week-old YAP/FBXL5 liver-dKO mice compared with that of the controls (Fig. 5 C). These results combined suggested that hepatic iron accumulation promotes bile acid-induced ferroptosis, thereby contributing to liver injury in cholestatic liver disease. Low-iron diet and antioxidant treatment suppress liver injury in YAP/FBXL5 liver-dKO mice Given that ferroptosis inhibitors suppress bile acid-induced cell death ex vivo , we next examined whether ferroptosis inhibition through iron restriction or antioxidant administration could ameliorate liver injury in vivo . To assess the effect of iron restriction, we implemented a dietary intervention in YAP/FBXL5 liver-dKO neonates by switching lactating mothers from a standard to an iron-deficient diet immediately after birth, thereby limiting iron transfer through breast milk until postnatal day 14 (Fig. 6 A). This intervention significantly reduced serum ALT levels in YAP/FBXL5 liver-dKO pups (Fig. 6 B). Histological analysis further revealed a marked decrease in hepatocyte death in iron-restricted mice compared to that in mice maintained on an iron-sufficient diet (Fig. 6 C), indicating that reduced iron availability alleviated liver injury. To evaluate the therapeutic potential of antioxidant treatment, we administered N-acetyl-L-cysteine (NAC), a glutathione precursor and ROS scavenger, via the drinking water of nursing mothers from birth to 4 weeks of age (Fig. 6 D). NAC treatment reduced in serum ALT levels (Fig. 6 E) and mitigated hepatocyte death (Fig. 6 F) in YAP/FBXL5 liver-dKO mice. Together, these findings supported a model in which hepatic iron overload drives ferroptosis during cholestasis, thereby exacerbating liver injury. Targeting ferroptosis through iron restriction or antioxidant therapy effectively mitigated hepatocyte death and liver damage in YAP/FBXL5 liver-dKO mice. DISCUSSION Several in vitro studies have shown that hydrophobic bile acids directly induce the apoptosis of primary hepatocytes 27 , 28 . In this context, oxidative stress has been proposed as a driving factor in the induction of apoptosis, with mitochondria-derived ROS (mtROS) generated by hydrophobic bile acids being implicated as a primary mechanism 27 , 28 . Studies have indicated that hydrophobic bile acids induce a mitochondrial permeability transition, causing the collapse of the mitochondrial membrane potential 29 , rupture of the outer mitochondrial membrane, and generation of ROS 30 . Increased ROS levels are suggested to affect downstream mitochondrial proteases 31 , BAX translocation 28 , and the release of cytochrome c 32 , thereby inducing apoptosis. However, disruption of the mitochondrial membrane potential by bile acids has also been implicated in mechanisms leading to necrosis 33 , 34 , suggesting that bile acid-induced cytotoxicity encompasses multiple forms of cell death beyond apoptosis. In this study, we demonstrated that iron sensitizes liver tissues to ferroptosis under hydrophobic bile acid exposure in an ex vivo culture system. mtROS is proposed to react with iron through Fenton reaction, resulting in hydroxyl radical production and lipid peroxidation 35 , 36 . Therefore, bile acids-induced mtROS generation may enhance the susceptibility of cholestatic livers to ferroptosis under iron overload. In the intestinal tract of high fat diet-fed mice, bile acid has been suggested to promote iron uptake through the induction of HIF2a expression 37 . However, it remains unclear whether bile acids enhance iron uptake in the liver under conditions of cholestatic liver disease. Further investigations are required to clarify whether bile acids orchestrate the regulation of both iron metabolism and ROS generation or induce ferroptosis under conditions where iron accumulation occurs as a secondary effect of etiologies, such as inflammation. Previous studies have investigated the pathogenic roles of apoptosis and necroptosis in cholestatic liver disease progression. In patients with primary biliary cirrhosis (PBC), upregulation of the FAS ligand, which promotes apoptosis 38 , 39 , and enhanced mixed lineage kinase domain-like protein (MLKL) phosphorylation, which signals necroptosis 40 , have been observed. Apoptosis has consistently been considered a pivotal contributor to liver injury and subsequent progression of liver fibrosis in the context of cholestatic liver disease. Indeed, liver injury and fibrosis induced by bile duct ligation (BDL) were attenuated in Fas-deficient lpr mice compared to wild-type mice 41 . The mechanism underlying apoptosis-mediated disease progression suggests that the phagocytosis of apoptotic bodies by hepatic stellate cells promotes pro-fibrotic activation and fibrogenesis 42 , 43 . Several lines of evidence suggest that necroptosis exerts pro-inflammatory and pro-fibrotic effects in various inflammatory conditions, including liver diseases. However, a previous study showed that BDL-induced apoptosis and liver fibrosis were comparable between mice deficient in RIP3, which is a key regulator of necroptosis, and wild-type mice 40 . Therefore, the role of necroptosis in the progression of cholestatic liver disease remains unclear. In our study, we observed substantial worsening of liver fibrosis in a model of cholestatic liver disease associated with iron overload, suggesting that ferroptosis plays a role in the progression of liver fibrosis. Supporting this observation, the induction of ferroptosis in an iron overload model mouse has been demonstrated to accelerate liver fibrosis 44 . This finding implies that, in addition to apoptosis, ferroptosis is a key pathogenic event during the progression of cholestatic liver injury. However, the molecular mechanisms through which ferroptosis promotes liver fibrosis require further investigation. In biliary atresia, inflammation and oxidative stress are prominently observed 45 . Emerging evidence suggests that ferroptosis exacerbates inflammation through immunogenicity 37 , 46 , 47 . This evidence also implies that ferroptosis is critical in facilitating the progression of chronic pathological states in cholestatic liver disease. Oxidative stress, a hallmark of apoptosis, has been reported to induce the upregulation of heme oxygenase 1 (HO-1), which, in turn, has been suggested to contribute to iron accumulation 40 . Given that iron accumulation appears to be a secondary effect of oxidative stress, ferroptosis may be considered as a later exacerbating event following apoptosis. However, the exact roles of apoptosis and ferroptosis in disease progression are not fully understood. Apoptosis and ferroptosis are likely interconnected and contribute to the disease progression. Therefore, it is essential to investigate the mechanisms that link these processes in future studies. Pediatric cholestatic liver diseases lack well-defined therapeutic approaches because the factors that contribute to their pathogenesis and progression are unclear. This study highlights the role of iron as an exacerbating factor in pediatric cholestatic liver diseases and suggests that targeting ferroptosis could be a promising strategy for controlling the progression of these diseases. Currently, ursodeoxycholic acid (UDCA), which exerts anti-apoptotic effects, is used as a treatment for cholestatic liver disease, but its used is limited by the delay in the time to liver transplantation or death 48 . Our findings indicated that beyond apoptosis, iron and ferroptosis could serve as important and potential therapeutic targets. Furthermore, bile acids are widely involved in metabolism 49 , immunity 50 , 51 , and various other processes 52 . The relationship between bile acids and ferroptosis identified in this study could have implications in these areas, suggesting its potential application in various diseases. MATERIALS AND METHODS Animals Yap flox/flox (Stock #030532) 53 and Alb -Cre (Stock #003574) 54 were obtained from Jackson Laboratory. Fbxl5 flox/flox mice were generated as previously described 18 . These lines were then crossed to generate Alb -Cre/ Yap flox/flox and Alb -Cre/ Yap flox/flox / Fbxl5 flox/flox mice. All mice were backcrossed to a C57BL/6 background for more than seven generations and maintained under specific pathogen-free conditions. Animals were group-housed in temperature-controlled conditions (22°C ± 2°C) with a 12-h light/dark cycle and had ad libitum access to food and water. Unless otherwise noted, experiments were conducted using male mice aged 2–10 weeks. Yap flox/flox and Yap flox/flox / Fbxl5 flox/flox mice were used as controls. For the low-iron diet intervention, mother mice were transitioned from a standard diet (CA-1 containing Fe; 30 mg/100 g feed) (CLEA, Tokyo, Japan) to a low-iron diet (AIN-93G containing Fe; 0.32 mg/100 g feed; CLEA) at the time of delivery and allowed ad libitum access to the low-iron diet for 2 weeks. For the NAC treatment, the mothers were switched from standard water (filtered and UV-irradiated tap water) to NAC water (2 g/L, filtered) (Wako, #1705131), which was provided ad libitum for 4 weeks. All mouse experiments were approved by the Animal Ethics Committee of Kumamoto University and the Institute of Science, Tokyo. Histological analysis of mouse liver To prepare paraffin sections, liver tissues were harvested and fixed immediately in 10% neutral-buffered formalin (Wako, 062-01661) at room temperature overnight. After fixation, liver sections were dehydrated, embedded in paraffin, and cut into 5 µm sections using a microtome (Leica RM2125). The sections were deparaffinized and rehydrated before staining. For Sirius red staining, paraffin sections were stained with Sirius red solution [1% Sirius red (Muto Pure Chemicals, #3306-1): Ven Gieson Solution P (Wako, #224–01405) = 1:30] for 1 h, followed by washing with 0.01 N HCl (Wako, #080-01066) for 1 min. For hematoxylin-eosin (HE) staining, paraffin sections were stained with hematoxylin (Sakura Finetek Japan, #9131-2)and eosin (Sakura Finetek Japan, #9135). For DAB-enhanced Perls staining, paraffin sections were incubated with buffer containing 2% potassium ferrocyanide (II) trihydrate (Wako, 167–03722) and 0.4% HCl (Wako, #080-01066) for 30 min, followed by development with DAB using a Peroxidase Stain DAB kit (Nacalai Tesque, #23985-50). The sections were dehydrated in xylene after each staining, and coverslips were applied using Mount-Quick (DAIDO SANGYO, #DM-01). Sections were imaged and analyzed using a KEYENCE BZ-X800 all-in-one microscope (KEYENCE). Immunostaining Paraffin sections (5 µm) were deparaffinized and rehydrated. Sections were subjected to antigen retrieval by heating in citrate buffer pH 6 [10 mM trisodium citrate dihydrate (Wako, #191–01785) solution and 10 mM citric acid monohydrate (Wako, #038-03505)] in a microwave (600 W) for 10 min. For immunohistochemistry, sections were subjected to 0.3% hydrogen peroxide (Wako, #081-04215) in methanol for 30 min. After two times washing with dH2O, sections were permeabilized with 0.2% Triton X-100 (polyoxyethylene (10) octylphenyl ether, Wako, #168-11805) in phosphate-buffered saline (PBS) for 10 min. Sections were then washed three times with PBS, followed by blocking with 5% goat serum (Sigma-Aldrich, #G9023-10ML) for 60 min. Sections were washed with PBS and then incubated with a primary antibody in PBS containing 1% bovine serum albumin (BSA) (Wako, #018-15154) at 4°C overnight. After incubation, the sections were washed three times with PBS, followed by sensitization with the VECTASTAIN Elite ABC-HRP kit and peroxidase (Rabbit IgG) (Vector Laboratories, #PK6101) for 30 min. After washing three times, the color was developed using a Peroxidase Stain DAB kit (Nacalai Tesque, #23985-50). Sections were counterstained with hematoxylin and dehydrated with xylene, and coverslips were applied using Mount-Quick (DAIDO SANGYO, #DM-01). For immunofluorescence staining, the sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate buffer, as described above. Thereafter, sections were permeabilized with 0.2% Triton X-100 in PBS for 10 min, followed by blocking with 5% goat serum for 60 min. Sections were then washed and incubated with primary antibody in PBS containing 1% BSA, 0.2% Triton-100 at 4°C overnight. Following three washes, sections were incubated with secondary antibody and DAPI (DOJINDO, #D523) in PBS containing 0.2% Triton X-100 for 60 min at room temperature in the dark. The sections were washed three times, mounted, and coverslipped using Fluoromount (COSMO BIO, K024). The images were acquired using a KEYENCE BZ-X800 all-in-one microscope (KEYENCE). The following antibodies were used: cytokeratin 19 (1:2000 dilution) (Abcam #ab133496), 4-hydroxyneonal (4-HNE) (1:1000 dilution) (Abcam #ab46545), and Alexa Fluor 546-labeled anti-rabbit IgG secondary antibody (1:500 dilution) (Invitrogen #A11030). Total non-heme iron measurement Frozen liver tissues were thawed to room temperature and washed three times (30 min/each) with PBS, followed by incubation at 45°C for 48 h. The dried tissues were finely crushed and added to an acidic solution [0.6 mM trichloroacetic acid (Nacalai Tesque, #34605-24), 14% HCl (Wako, #080-01066)] and incubated at 65°C for 48 h. Samples were centrifuged at 1,500 × g for 15 min. The resulting supernatant was mixed with chromogen solution [0.01% bathophenanthroline disulfonic acid disodium salt (Wako, 348 − 00191), 0.1% mercaptoacetic acid (Nacalai Tesque, #33711-12), and 3.5 M sodium acetate (Wako, #198-15965)], followed by incubation at room temperature for 10 min. The absorbance of each sample was then measured at 535 nm. A dilution series of iron standard solutions (Nacalai Tesque, #37514-24) was prepared, and their absorbance was measured similarly to generate a standard curve. Serum analysis Serum alanine aminotransferase (ALT), ALP, and bilirubin (total, conjugated, and unconjugated) levels were measured using a standard clinical autoanalyzer (SRL, Inc.). Precision-cut liver slice (PCLS) culture The liver was excised from the mouse, placed in Kreb’s buffer [2.0 g/L D-glucose (Wako, #049-31165), 0.288 g/L magnesium sulfate (Nacalai Tesque, #137–00402), 0.16 g/L KH 2 PO 4 (Wako, #169–04245), 0.35 g/L KCl (Wako, #163–03545), 6.9 g/L NaCl (Wako, #195–01663), 0.282 g/L CaCl 2 (Wako, #163–03545), and 2.1 g/L NaHCO 3 (Wako, #191–01305); pH 7.4] and kept on ice until slicing. The liver lobe was isolated and sliced into 200 µm thickness with a vibratome (Neo LinerSlicer M, DOSAKA EM CO., LTD). The sections were then immediately placed in a 12-well plate containing 250 µL of DMEM supplemented 10% fetal bovine serum (FBS) and incubated at 37°C in 5% CO 2 . Measurement of bile acid toxicity in PCLS The medium of the PCLS was replaced with a deoxycholic acid (DCA) [sodium deoxycholate (Wako, #192–08312) in dH2O] dissolved in DMEM containing 10% FBS, followed by incubation at 37°C in 5% CO 2 for 24 h. In several experiments, ammonium iron (III) citrate (FAC); Sigma-Aldrich, #F 5879-100G), deferoxamine (DFO) (Abcam, #ab120727), and ferrostatin-1(Fer-1) (Cayman, #17729) were added simultaneously. After incubation, PCLSs were stained with Hoechst33342 (1 mg/mL) (Sigma-Aldrich, #B2261-25G) and PI (1:1000) (DOJINDO, #341–07881) for 30 min in 37°C in 5% CO 2 . Following a PBS wash, PCLSs were fixed using 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature in the dark. The PCLSs were washed three times, mounted, and coverslipped with Fluoromount. Images were captured using a confocal microscope (FV3000; Evident). Quantification of PI and Hoechst staining was performed using Fiji/ImageJ software. Ink injection for bile duct visualization Mice were euthanized, followed by a laparotomy and clipping of the common bile duct. Subsequently, a 29G insulin syringe (TERUMO, #SS-10M2913A) was inserted into the gallbladder, and pigment-based ink (KIWAGURO, SAILOR) was gradually infused to stain the bile ducts retrogradely. After staining, the livers were incubated in 10%, 40%, 80% ethanol in PBS on a gentle rocker at 4°C, each for 1 h, followed by overnight dehydration in 100% ethanol. Following dehydration, BABB solution [benzyl alcohol (Wako, #024-01286):benzyl benzoate (Wako, #025-01336) = 1:2] was used to clear the liver by rocking it at 4°C until clearing. Images were captured using a stereoscopic microscope (M165FC; Leica). Immunoblot analysis Liver tissues were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100) with protease inhibitors [10 µM Leupeptin (Wako, #336-40413), 1 µM Pepstatin A (Wako, #330-43973) and 1 mM PMSF (Wako, #160-12183)] on ice. After homogenization, samples were kept on ice for 30 min, followed by centrifugation at 12,000 × g for 20 min at 4°C. The resulting supernatant was used for analysis. Protein volume was measured by TaKaRa Bradford Protein Assay Kit (Takara, #T9310A), and equal amounts of protein were separated using an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by transfer onto PVDF membrane (Millipore, #IPVH00010). The membrane was blocked with 5% skim milk (Wako, #190-12865) for 30 min at room temperature, and immunoblotted with primary antibodies in Tris-buffered saline (TBS) containing 0.1% Tween20 and 0.5% skim milk at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 60 min. Proteins were developed using a chemiluminescent HRP substrate (Millipore, WBKLS0500), and images were captured using a ChemiDoc Touch imaging system (Bio-Rad Laboratories, #17001401JA). The following antibodies were used in the experiments: HRP-conjugated anti-YAP (1:2000 dilution) (Cell Signaling Technology, #15208), anti-IRP2 (1:2000 dilution) (Millipore, #MABS2020-100UG), HSP90 (1:2000) (BD Transduction, #610418), and HRP-conjugated anti-mouse IgG (Cell Signaling Technology, #7026) were used. Quantification and statistical analysis Quantitative results are shown as the mean ± SEM. All statistical analyses were performed using GraphPad Prism 10 software (GraphPad Software, Inc.) according to the methods described in the figure legends. Statistical significance was set at P < 0.05. Declarations OTHER NOTES Acknowledgements The authors thank the Center for Animal Resources and Development, Kumamoto University, for their support in animal experiments; the Core Laboratory for Medical Research and Education, Kumamoto University School of Medicine, for their technical support; and Hiroyuki Oshiumi for discussions. Conflict of interests The authors declare no competing interests. Author contributions Conceptualization: Y.O, Y.K, and T.Moroishi; Methodology: Y.O, Y.K, A.M, A.N, T.Matsumoto, K.I.N and T.Moroishi; Investigation: Y.O, Y.K, A.M, M.F.S, and T.Moroishi; Writing of original draft: Y.O, Y.K, and T.Moroishi; Funding acquisition: Y.K, T,M; Supervision: T.Moroishi. All the authors have read and approved the final manuscript. Ethics The animal study design was approved by the Animal Committee of Kumamoto University (A2023-031) and Institute of Science Tokyo (A2024-166C). Funding This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants (23K19098, 24H00864, and 24H00865 to TM; 22K15396 and 24K10094 to Y.K), the Japan Science and Technology Agency (JST) FOREST Program (JPMJFR226J to T.M), JST SPRING (JPMJSP2127 to Y.O), and the Kato Memorial Bioscience Foundation (to T.M). References Sutton, H., Karpen, S. J. & Kamath, B. M. Pediatric Cholestatic Diseases: Common and Unique Pathogenic Mechanisms. Annu Rev Pathol 19 , 319-344, doi:10.1146/annurev-pathmechdis-031521-025623 (2024). Elisofon, S. A. et al. Society of pediatric liver transplantation: Current registry status 2011-2018. Pediatr Transplant 24 , e13605, doi:10.1111/petr.13605 (2020). Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149 , 1060-1072, doi:10.1016/j.cell.2012.03.042 (2012). Zhang, N. et al. The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals. Dev. 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Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J. Biol. Chem. 274 , 305-315, doi:10.1074/jbc.274.1.305 (1999). Additional Declarations (Not answered) Supplementary Files ohtasup250515.docx Supplemental Figure Legends ohtafigs1250509.pdf Supplemental Figure 1 ohtafigs2250509.pdf Supplemental Figure 2 ohtafigs3250507.pdf Supplemental Figure 3 Uncroppedoriginalwesternblots250502.pdf Original western blot images Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6671814","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":459764137,"identity":"b7ad24d8-a223-48f4-b9a0-6c1616f92c70","order_by":0,"name":"Toshiro Moroishi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYLCCBIYDPCCamcGAgYEfyJAA8RgbCGlhg2qRbCNGCwPDAQaIFiAwOAbVggvotvc+fPDgzx0ZBvnmw58LCu7JGd/vTrzBUGPHwDwbuzVmZ44bGyS2PQM6jC1NeoZBsbHZMd7NFgzHkhkY5xzAruVGGptEYsNhoBYeM2Yeg4TEbcd4t0kwsB1gYJyRgFtLwh+wFuPPIC2b20Ba/hHSwgbWYiAN0rKBDaiFsQ2PljPHmIF+OcwD9EoaSIuxxLHczRaJfck8OP1yvI3x4Y8/h+35mQ8f/szzJ0GOv/nsxhsfvtnJGeIIMThgQ+EBncRjOAO/DixAHm+EjoJRMApGwQgCANMhVrW7rKMTAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6419-3882","institution":"Institute od Science Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Toshiro","middleName":"","lastName":"Moroishi","suffix":""},{"id":459764138,"identity":"028ac350-c9a9-4554-b5b2-a8b855f1becf","order_by":1,"name":"Yudai OHTA","email":"","orcid":"","institution":"Institute od Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Yudai","middleName":"","lastName":"OHTA","suffix":""},{"id":459764139,"identity":"074219d7-14e8-4018-bad3-6b58267e100f","order_by":2,"name":"Yohei KANAMORI","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Yohei","middleName":"","lastName":"KANAMORI","suffix":""},{"id":459764140,"identity":"90cef31f-7801-4382-81e2-ca5cae5864eb","order_by":3,"name":"Ayato MAEDA","email":"","orcid":"","institution":"Institute od Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Ayato","middleName":"","lastName":"MAEDA","suffix":""},{"id":459764141,"identity":"5356d4ab-047b-451a-a885-50ed8ff76467","order_by":4,"name":"Mohamed Fathi Saleh","email":"","orcid":"","institution":"Tanta University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"Fathi","lastName":"Saleh","suffix":""},{"id":459764142,"identity":"0f712194-06a3-44ab-914e-2dddc2d21c37","order_by":5,"name":"Akihiro NITA","email":"","orcid":"","institution":"Institute od Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"NITA","suffix":""},{"id":459764143,"identity":"74866b7c-f315-4599-bf5d-958317e5f96e","order_by":6,"name":"Takashi MATSHMOTO","email":"","orcid":"https://orcid.org/0000-0001-6791-6441","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"MATSHMOTO","suffix":""},{"id":459764144,"identity":"42061333-aa43-4122-a56e-ef3e14be947f","order_by":7,"name":"Keiichi I Nakayama","email":"","orcid":"","institution":"Tokyo Medical and Dental University","correspondingAuthor":false,"prefix":"","firstName":"Keiichi","middleName":"I","lastName":"Nakayama","suffix":""}],"badges":[],"createdAt":"2025-05-15 10:51:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6671814/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6671814/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83357447,"identity":"4209ea87-e69a-47f8-bc60-6f5511103eb9","added_by":"auto","created_at":"2025-05-23 15:25:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2836443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHepatic iron levels increased in mice with YAP deficiency-induced cholestatic liver injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp; Non-heme iron content in the livers of control (\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e; n = 10) and YAP liver-KO (knockout) (\u003cem\u003eAlb\u003c/em\u003e-Cre/\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e; n = 12) mice at 10 weeks of age. Data are presented as the mean ± SEM. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 (unpaired two-tailed Student’s\u003cem\u003e t-\u003c/em\u003etest).\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp; Representative histological images showing hepatic iron distribution in control and YAP liver-KO mice, visualized by DAB-enhanced Perls’ staining. Lower panels show higher magnification of the corresponding upper panels. Scale bar, 50 µm.\u003c/p\u003e\n\u003cp\u003e(C)\u0026nbsp; Correlation between non-heme iron content and serum total bilirubin levels in 10-week-old YAP liver-KO mice (n = 12).\u003c/p\u003e","description":"","filename":"ohtafig1250509.png","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/8575f9bec0ffdcfce17062e6.png"},{"id":83356260,"identity":"c6a3a097-d00d-4344-beb8-6d13b1958edb","added_by":"auto","created_at":"2025-05-23 15:09:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4819531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetically induced iron overload exacerbates cholestatic liver disease progression in YAP-deficient mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative histological images showing hepatic iron distribution in control, FBXL5 liver-KO (\u003cem\u003eAlb\u003c/em\u003e-Cre/\u003cem\u003eFbxl5\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e), YAP liver-KO (\u003cem\u003eAlb\u003c/em\u003e-Cre/\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e), and YAP/FBXL5 liver-dKO (double knockout) (\u003cem\u003eAlb\u003c/em\u003e-Cre/\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e/\u003cem\u003eFbxl5\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e) mice at 10-weeks of age, visualized using DAB-enhanced Perls’ staining. Scale bar, 50 µm.\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp; Comparison of jaundice-like appearance in mouse limbs. Scale bar, 4 mm.\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp; Serum levels of total (left) and conjugated (right) bilirubin in control (n = 10), FBXL5 liver-KO (n = 5), YAP liver-KO\u003cem\u003e \u003c/em\u003e(n = 13), and YAP/FBXL5 liver-dKO (n = 6) mice at 10 weeks of age. Data are presented as the mean ± SEM. ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(C)\u0026nbsp; Liver fibrosis visualized by Sirius Red staining (left), with quantification of Sirius Red–positive area normalized to the total hepatic area (right). Scale bar, 50 µm. Control (n = 3), FBXL5 liver-KO (n = 4), YAP liver-KO (n = 7), and YAP/FBXL5 liver-dKO (n = 6) mice were analyzed at 10 weeks of age. Data are presented as the mean ± SEM. ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 (One-way ANOVA followed by Tukey’s multiple comparison test).\u003c/p\u003e","description":"","filename":"ohtafig2250509.png","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/918baa020c4ff78cdcb0256e.png"},{"id":83355070,"identity":"44fdc786-8e8a-4008-ac47-816224dedf50","added_by":"auto","created_at":"2025-05-23 15:01:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2876523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIron overload enhances liver injury in YAP-deficient mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Time-course of serum total bilirubin levels in control (n = 5–15), FBXL5 liver-KO (n = 3), YAP liver-KO (n = 4–12), and YAP/FBXL5 liver-dKO (n = 6–12) mice at 2, 4, and 8 weeks of age. Data are presented as the mean ± SEM.\u003c/p\u003e\n\u003cp\u003e(B) Serum levels of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in control (n = 11), FBXL5 liver-KO (n = 3), YAP liver-KO (n = 6–12), and YAP/FBXL5 liver-dKO\u003cem\u003e \u003c/em\u003e(n = 8–11) at 2 weeks of age. Data are presented as the mean ± SEM. ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test).\u003c/p\u003e\n\u003cp\u003e(C) Hepatic cell death visualized by hematoxylin and eosin (H\u0026amp;E) staining (left), with quantification of the cell death area normalized to the total hepatic area (right). Asterisks indicate the representative areas of cell death. Scale bar, 100 µm. Control (n = 5), FBXL5 liver-KO (n = 3), YAP liver-KO (n = 6), and YAP/FBXL5 liver-dKO (n = 6) mice were analyzed at 2 weeks of age. Data are presented as the mean ±SEM. ns, not significant; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test).\u003c/p\u003e","description":"","filename":"ohtafig3250503.png","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/201c7a18119229c59f374c41.png"},{"id":83355069,"identity":"a96ce557-bf62-42a4-8dc8-a466204dcecd","added_by":"auto","created_at":"2025-05-23 15:01:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2558744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIron increases susceptibility to bile acid toxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp; Schematic of the experimental procedure using precision-cut liver slices (PCLS).\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp; Representative images (left) showing propidium iodide (PI) and Hoechst staining of PCLS following exposure to 750 μM deoxycholic acid (DCA). Quantification (right) shows the ratio of PI-positive to Hoechst-positive cells. Scale bar, 25 μm. PCLS were prepared from control (n = 3), FBXL5 liver-KO (n = 4), YAP liver-KO (n = 4), and YAP/FBXL5 liver-dKO (n = 3) mice at 2 weeks of age. Data are presented as the mean ± SEM. ns, not significant; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test).\u003c/p\u003e\n\u003cp\u003e(C)\u0026nbsp; Cell viability of control PCLS after treatment with DCA (750 μM) and ferric ammonium citrate (FAC, 50 μg/mL) for 24 h. Representative images (left) and quantification of PI\u003csup\u003e+\u003c/sup\u003e/Hoechst\u003csup\u003e+\u003c/sup\u003e cells (right) are shown. Scale bar, 25 μm. n = 3 PCLS from 3 independent mice. Data are presented as the mean ± SEM. ns, not significant; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test).\u0026nbsp;\u003c/p\u003e","description":"","filename":"ohtafig4250510.png","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/2f2b711eca80f3171000d835.png"},{"id":83356699,"identity":"f320bd5a-f312-49cf-aa44-a80d498dd96e","added_by":"auto","created_at":"2025-05-23 15:17:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2686850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eToxic bile acid induces ferroptosis in iron-loaded cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images (left) and quantification (right) of FBXL5-deficient PCLS treated with DCA (750 μM) and deferoxamine (DFO, 100 μM) for 24 h. Scale bar, 25 μm. n = 3 PCLS from 3 independent mice. Data are presented as the mean ± SEM. ns, not significant; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test).\u003c/p\u003e\n\u003cp\u003e(B) Representative images (left) and quantification (right) of FBXL5-deficient PCLS treated with DCA (750 μM) and ferrostatin-1 (Fer-1, 10 µM) for 24 h. Scale bar, 25 μm. n = 3 PCLS from 3 independent mice. Data are presented as the mean ± SEM. ns, not significant; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test).\u003c/p\u003e\n\u003cp\u003e(C) Immunostaining for 4-hydroxynonenal (4-HNE, red) and DAPI (blue) in liver tissue from control and YAP/FBXL5 liver-dKO mice at 2 weeks of age. Scale bar, 50 µm.\u003c/p\u003e","description":"","filename":"ohtafig5250503.png","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/3d56e36cebb197a7942ba286.png"},{"id":83355080,"identity":"384e33cb-b114-4b0f-8671-00f472aeb4c2","added_by":"auto","created_at":"2025-05-23 15:01:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3557404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow-iron diet and antioxidant treatment suppress liver injury in YAP/FBXL5 liver-dKO mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration of the low-iron diet intervention. Lactating mothers were switched to a low-iron diet for two weeks following delivery.\u003c/p\u003e\n\u003cp\u003e(B) Serum ALT levels in control and YAP/FBXL5 liver-dKO mice on standard or low-iron diets. Control: standard (n = 4), low-iron (n = 6); YAP/FBXL5 liver-dKO: standard (n = 4), low-iron (n = 4). Data are presented as the mean ± SEM. ns, not significant; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (One-way ANOVA followed by Tukey’s multiple comparison test).\u003c/p\u003e\n\u003cp\u003e(C) Hepatic cell death visualized by H\u0026amp;E staining (left), with quantification of cell death area normalized to total hepatic area (right). Asterisks indicate representative areas of cell death. Scale bar, 50 µm. YAP/FBXL5 liver-dKO mice fed with standard (n = 4) or low-iron diet (n = 4) were analyzed at 2 weeks of age. Data are presented as the mean ±SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (unpaired two-tailed Student’s\u003cem\u003e t-\u003c/em\u003etest).\u003c/p\u003e\n\u003cp\u003e(D) Schematic illustration of the \u003cem\u003eN-\u003c/em\u003eacetyl\u003cem\u003e-L\u003c/em\u003e-cysteine (NAC) treatment. Lactating mothers received NAC (2 g/L) in drinking water for four weeks postpartum.\u003c/p\u003e\n\u003cp\u003e(E) Serum ALT levels in control and YAP/FBXL5 liver-dKO mice under standard conditions or NAC treatment at 4 weeks of age. Control: regular water (n = 6), NAC (n = 5); YAP/FBXL5 liver-dKO: regular water (n = 7), NAC (n = 5). Data are presented as the mean ±SEM. ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test).\u003c/p\u003e\n\u003cp\u003e(F) Hepatic cell death visualized by H\u0026amp;E staining (left), with quantification of cell death area normalized to total hepatic area (right). Asterisks indicate representative areas of cell death. Scale bar, 50 µm. YAP/FBXL5 liver-dKO mice treated with regular water or (n = 4) NAC (n = 5) were analyzed at 24weeks of age. Data are presented as the mean ±SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (unpaired two-tailed Student’s\u003cem\u003e t-\u003c/em\u003etest).\u003c/p\u003e","description":"","filename":"ohtafig6250510.png","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/bf500c4c05d55b40ed6b79da.png"},{"id":83357580,"identity":"c52ab3d2-08ac-41d4-b244-df7c3bea76e5","added_by":"auto","created_at":"2025-05-23 15:33:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21996674,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/c65ac055-0e38-4ee4-a41e-8b6d0accd69c.pdf"},{"id":83355060,"identity":"e9fab18a-19d3-4dc4-b805-66bfbc225e8c","added_by":"auto","created_at":"2025-05-23 15:01:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21394,"visible":true,"origin":"","legend":"Supplemental Figure Legends","description":"","filename":"ohtasup250515.docx","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/341484dcf681d1fa4e405a35.docx"},{"id":83355065,"identity":"b13a831a-f4f2-4c55-813f-33385d56dc8f","added_by":"auto","created_at":"2025-05-23 15:01:07","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":703063,"visible":true,"origin":"","legend":"Supplemental Figure 1","description":"","filename":"ohtafigs1250509.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/dc25c9b461ca408343d73f65.pdf"},{"id":83355061,"identity":"296fbd7b-f6fb-40b0-9757-0a016a7c2de8","added_by":"auto","created_at":"2025-05-23 15:01:07","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":538862,"visible":true,"origin":"","legend":"Supplemental Figure 2","description":"","filename":"ohtafigs2250509.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/52579c642558fc24d33f3580.pdf"},{"id":83355068,"identity":"fd03b749-79f4-4df6-a21e-746c9677eada","added_by":"auto","created_at":"2025-05-23 15:01:07","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":2072638,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental Figure 3\u003c/p\u003e","description":"","filename":"ohtafigs3250507.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/306f997a805c5c04572e747e.pdf"},{"id":83356257,"identity":"99676c15-5348-4dcb-af0c-d2e4ecd7a37f","added_by":"auto","created_at":"2025-05-23 15:09:07","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":583395,"visible":true,"origin":"","legend":"\u003cp\u003eOriginal western blot images\u003c/p\u003e","description":"","filename":"Uncroppedoriginalwesternblots250502.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6671814/v1/a5e8b4988514a27b2be48eed.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Iron exacerbates congenital cholestatic liver injury via bile acid-induced ferroptosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCholestatic liver diseases in the neonatal period typically present with cholestasis as an initial symptom, accompanied by inflammation and liver fibrosis, which ultimately progress to liver cirrhosis in severe cases. Congenital disorders associated with bile duct malformations, such as biliary atresia and Alagille syndrome, represent the most common causes of pediatric cholestasis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Approximately half of modern pediatric liver transplantations are related to cholestatic liver diseases\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and medical treatments for these conditions remain insufficiently established.\u003c/p\u003e \u003cp\u003eIntrahepatic bile ducts in mice originate from hepatoblasts, the common progenitors of the hepatocyte and cholangiocyte lineages, and differentiate between embryonic day 11.5 and 13.5. Several studies have indicated that Yes-associated protein (YAP), a transcriptional coactivator in the Hippo intracellular signaling pathway\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, regulates this differentiation process\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. YAP is widely recognized as a critical determinant of cell fate, modulating both cell differentiation and proliferation in diverse contexts, including cancer progression and embryonic development\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. During liver development, YAP has been suggested to promote the differentiation of hepatoblasts into cholangiocytes through interactions with SRY-box transcription factor 9 (SOX9) and the Notch signaling pathway\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Indeed, the loss of YAP during liver development leads to defects of biliary ductal structures and a marked paucity of bile ducts, causing long-term cholestatic injury\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although the underlying causes and pathological mechanisms of cholestasis in pediatric cholestatic liver disease are diverse, obstruction of bile flow and subsequent accumulation of intrahepatic bile components are common features. However, the detailed mechanisms underlying the progression of pediatric cholestatic liver disease remain unclear.\u003c/p\u003e \u003cp\u003eIron overload is a risk factor for worsening disease outcomes in various chronic liver diseases, such as hepatitis C virus infection (HCV)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, alcoholic liver disease\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and metabolic dysfunction-associated steatohepatitis (MASH)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Iron overload under pathological conditions leads to an imbalance in iron homeostasis and causes excessive ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e) accumulation. Fe\u003csup\u003e2+\u003c/sup\u003e facilitates reactive oxygen species (ROS) production through the Fenton reaction, resulting in lipid peroxidation and ultimately triggering ferroptosis, a recently identified form of regulated cell death distinct from apoptosis and necrosis, characterized by iron-dependent lipid peroxide accumulation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This process has been suggested to be a critical mechanism by which iron overload contributes to the progression of liver diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Despite its relevance in other liver disease, the involvement of ferroptosis in the pathogenesis of congenital cholestatic liver disease remains poorly understood.\u003c/p\u003e \u003cp\u003eCellular iron homeostasis is primarily regulated by the iron-sensing protein F-box and leucine-rich protein 5 (FBXL5) and the RNA-binding protein iron regulatory protein 2 (IRP2)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. IRP2 post-transcriptionally upregulates the expression of proteins responsible for iron uptake and simultaneously suppresses the expression of proteins involved in iron export, thereby increasing the cellular labile iron pool (Fe\u003csup\u003e2+\u003c/sup\u003e). FBXL5 is a substrate-recognition subunit of the SCF-type E3 ubiquitin ligase complex, facilitating the recruitment of IRP2 for its ubiquitination and subsequent degradation. FBXL5 binds directly to iron and is stabilized when intracellular iron is abundant, leading to the degradation of IRP2, which helps reduce cellular iron levels and maintain iron homeostasis\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. FBXL5 deficiency stabilizes IRP2 regardless of cellular iron levels, causing cellular iron overload. FBXL5-null mice display early embryonic lethality resulting from iron-induced oxidative stress, which is rescued by the additional deletion of IRP2, suggesting that the precise regulation of IRP2 by FBXL5 is crucial for maintaining cellular iron homeostasis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Additionally, studies using mice with a conditional deletion of FBXL5 in different cell types have demonstrated that FBXL5 deficiency leads to increased cellular iron levels, which is attributed to the aberrant accumulation of IRP2 \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we showed that hepatic iron accumulation correlates with cholestasis progression in mice with biliary malformations induced by hepatic YAP deficiency. Hepatic iron overload caused by FBXL5 depletion augmented congenital cholestatic liver disease phenotypes in hepatic YAP-deficient mice. Mechanistically, bile acid toxicity was exacerbated by ferroptosis, driven by iron accumulation, which accelerated liver injury and contributed to disease progression. This study provides insights into the role of iron as a pathogenic factor in congenital chronic cholestatic liver diseases.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHepatic iron accumulation is a hallmark of cholestatic liver disease induced by YAP deficiency\u003c/h2\u003e \u003cp\u003eTo investigate the pathogenesis of congenital cholestatic liver disease, we generated mice with liver-specific ablation of \u003cem\u003eYap\u003c/em\u003e by crossing \u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice with \u003cem\u003eAlbumin\u003c/em\u003e-Cre (\u003cem\u003eAlb\u003c/em\u003e-Cre) transgenic mice. Loss of YAP protein in the liver tissue was confirmed by immunoblotting (Supplementary Fig.\u0026nbsp;1A). The injection of India ink into the extrahepatic bile ducts of control (\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e) mice resulted in the flow of ink into intrahepatic bile ducts, allowing visualization of the interlobular bile ducts and bile canaliculi. In contrast, consistent with previous observations\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eAlb\u003c/em\u003e-Cre/\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e [hereafter referred to as YAP liver-KO (knockout)] mice showed no ink flow into the intrahepatic bile ducts (Supplementary Fig.\u0026nbsp;1B). Furthermore, immunostaining of biliary epithelial cells revealed well-organized tubular structures in control livers, whereas YAP-deficient livers exhibited obstructed duct-like structures (Supplementary Fig.\u0026nbsp;1C). Histological analysis revealed multiple areas of hepatic cell death in YAP-deficient livers (Supplementary Fig.\u0026nbsp;1C). These pathological features, including impaired bile flow, bile duct obstruction, and hepatocellular injury, demonstrated that YAP deletion during development recapitulates the key characteristics of congenital cholestatic liver disease. To explore potential pathogenic factors contributing to disease progression, we assessed hepatic iron levels in YAP liver-KO mice. Measurement of non-heme iron content using a bathophenanthroline-based colorimetric assay revealed significantly elevated hepatic iron levels in YAP liver-KO mice compared to those in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Diaminobenzidine (DAB)-enhanced Perls' staining further confirmed that iron accumulation was predominantly localized to the hepatocytes of YAP liver-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Notably, the degree of hepatic iron accumulation correlated with serum bilirubin levels in YAP liver-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), suggesting that iron deposition is associated with the progression of cholestatic liver disease in this model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenetically induced iron overload exacerbates cholestatic liver disease progression in YAP-deficient mice\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of iron in the pathologies of congenital cholestatic liver disease, we induced hepatic iron overload in YAP liver-KO mice by crossing them with \u003cem\u003eFbxl5\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice. Consistent with previous findings\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, FBXL5 deficiency led to the hepatic accumulation of IRP2 (Supplementary Fig.\u0026nbsp;2A) and subsequent iron overload (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) in both FBXL5 liver-knockout (\u003cem\u003eAlb\u003c/em\u003e-Cre\u003cem\u003e/Fbxl5\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e) and YAP/FBXL5 double knockout (\u003cem\u003eAlb\u003c/em\u003e-Cre/\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e/\u003cem\u003eFbxl5\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e) mice, hereafter referred to as FBXL5 liver-KO and YAP/FBXL5 liver-dKO mice, respectively. FBXL5 liver-KO mice displayed no signs of cholestatic liver disease, including jaundice, hyperbilirubinemia, or liver fibrosis. In contrast, YAP/FBXL5 liver-dKO mice exhibited more pronounced jaundice than YAP liver-KO mice at 10 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Serum analyses confirmed that cholestasis was significantly exacerbated in YAP/FBXL5 liver-dKO mice, as indicated by elevated total and conjugated bilirubin levels compared to those in YAP liver-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, Sirius red staining revealed enhanced liver fibrosis in YAP/FBXL5 liver-dKO mice compared to that in YAP liver-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results indicate that iron accumulation resulting from FBXL5 deficiency aggravates cholestatic liver injury and fibrosis in the context of YAP deficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIron overload enhances liver injury in YAP-deficient mice\u003c/h3\u003e\n\u003cp\u003eTo elucidate how iron accumulation contributes to the progression of congenital cholestatic liver disease, we analyzed temporal changes in the severity of cholestasis. From 2 to 8 weeks of age, total bilirubin levels were persistently elevated in both YAP liver-KO and YAP/FBXL5 liver-dKO mice compared to those in control and FBXL5 liver-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Notably, while bilirubin levels were comparable between YAP liver-KO and YAP/FBXL5 liver-dKO mice at 2 weeks, significantly higher levels were observed in dKO mice at 4 and 8 weeks. These observations suggested that pathological events occurring as early as 2 weeks of age may underlie iron overload-driven exacerbation of cholestatic liver disease in YAP-deficient livers. Given that liver injury caused by cholestasis can disrupt the intrahepatic bile ducts and accelerate disease progression\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, we assessed liver damage at 2 weeks of age. While serum alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were not significantly different among control, FBXL5 liver-KO, and YAP liver-KO mice, YAP/FBXL5 liver-dKO mice exhibited significantly elevated levels of both ALT and ALP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Histological analysis confirmed extensive hepatocyte death in the livers of YAP/FBXL5 liver-dKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results suggested that hepatic iron overload caused by FBXL5 deficiency aggravates early liver injury, thereby accelerating the progression of cholestatic liver disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIron increases susceptibility to bile acid toxicity\u003c/h3\u003e\n\u003cp\u003eTo investigate the molecular mechanism by which iron overload exacerbates liver injury in YAP-deficient livers, we examined the interplay between iron and bile acid toxicity. Previous studies have demonstrated that the cytotoxic effects of bile acids primarily drive hepatocellular and biliary injuries in cholestatic liver disease\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To assess how iron influences bile acid-induced cell death, we utilized \u003cem\u003eex vivo\u003c/em\u003e precision-cut liver slices (PCLS), a 3D culture system that preserves the native liver architecture, and stimulated them with deoxycholic acid (DCA)\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, a representative hydrophobic and toxic bile acid implicated in cholestatic liver injury. We first established a dose-response relationship using PCLS in control mice. Cell death, assessed using propidium iodide (PI) staining, was induced by DCA at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;1 mM, while concentrations\u0026thinsp;\u0026lt;\u0026thinsp;800 \u0026micro;M had minimal cytotoxic effects (Supplemental Figure. 3A). Based on this, we selected 750 \u0026micro;M DCA\u0026mdash;a sublethal dose for control tissue\u0026mdash;to assess the impact of iron overload on bile acid sensitivity. We then examined the response to PCLS in 2-week-old FBXL5 liver-KO, YAP liver-KO, and YAP/FBXL5 liver-dKO mice. Exposure to 750 \u0026micro;M DCA caused only mild cell death (~\u0026thinsp;20%) in YAP liver-KO slices, but resulted in marked cell death (\u0026gt;\u0026thinsp;60%) in PCLS from both FBXL5 liver-KO and YAP/FBXL5 liver-dKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Notably, the extent of DCA-induced cell death was similar between FBXL5 liver-KO and YAP/FBXL5 liver-dKO PCLS, indicating that iron overload due to FBXL5 deficiency, rather than YAP loss, is the primary factor sensitizing the liver tissue to bile acid toxicity. Furthermore, treatment of control PCLS with ferric ammonium citrate (FAC), an exogenous iron source, similarly enhanced DCA-induced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Collectively, these results suggested that hepatic iron overload enhances the cytotoxic effects of bile acids and contributes to the progression of liver injury in cholestatic liver disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eToxic bile acid induces ferroptosis in iron-loaded cells\u003c/h3\u003e\n\u003cp\u003eGiven that excess iron is a known driver of ferroptosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, we hypothesized that liver tissue with iron overload would exhibit increased susceptibility to ferroptosis. To test this, we evaluated whether bile acid-induced cell death involves ferroptosis in the context of iron overload. PCLS from FBXL5 liver-KO mice were exposed to DCA in the presence or absence of two established ferroptosis inhibitors\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e: the iron chelator deferoxamine (DFO) and the lipid peroxidation scavenger ferrostatin-1 (Fer-1). Consistent with previous observations, DCA exposure induced substantial cell death (\u0026gt;\u0026thinsp;60%) in PCLS from FBXL5-deficient mice. However, treatment with DFO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) or Fer-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) significantly reduced DCA-induced cell death, indicating that cytotoxicity depended on both iron and lipid peroxidation, which are hallmarks of ferroptosis. Supporting this interpretation, immunohistochemical analysis revealed marked accumulation of 4-hydroxynonenal (4-HNE), a well-established lipid peroxidation product and ferroptosis marker\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, in liver tissues from 2-week-old YAP/FBXL5 liver-dKO mice compared with that of the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results combined suggested that hepatic iron accumulation promotes bile acid-induced ferroptosis, thereby contributing to liver injury in cholestatic liver disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLow-iron diet and antioxidant treatment suppress liver injury in YAP/FBXL5 liver-dKO mice\u003c/h2\u003e \u003cp\u003eGiven that ferroptosis inhibitors suppress bile acid-induced cell death \u003cem\u003eex vivo\u003c/em\u003e, we next examined whether ferroptosis inhibition through iron restriction or antioxidant administration could ameliorate liver injury \u003cem\u003ein vivo\u003c/em\u003e. To assess the effect of iron restriction, we implemented a dietary intervention in YAP/FBXL5 liver-dKO neonates by switching lactating mothers from a standard to an iron-deficient diet immediately after birth, thereby limiting iron transfer through breast milk until postnatal day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This intervention significantly reduced serum ALT levels in YAP/FBXL5 liver-dKO pups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Histological analysis further revealed a marked decrease in hepatocyte death in iron-restricted mice compared to that in mice maintained on an iron-sufficient diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), indicating that reduced iron availability alleviated liver injury. To evaluate the therapeutic potential of antioxidant treatment, we administered N-acetyl-L-cysteine (NAC), a glutathione precursor and ROS scavenger, via the drinking water of nursing mothers from birth to 4 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). NAC treatment reduced in serum ALT levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and mitigated hepatocyte death (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) in YAP/FBXL5 liver-dKO mice. Together, these findings supported a model in which hepatic iron overload drives ferroptosis during cholestasis, thereby exacerbating liver injury. Targeting ferroptosis through iron restriction or antioxidant therapy effectively mitigated hepatocyte death and liver damage in YAP/FBXL5 liver-dKO mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSeveral \u003cem\u003ein vitro\u003c/em\u003e studies have shown that hydrophobic bile acids directly induce the apoptosis of primary hepatocytes\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In this context, oxidative stress has been proposed as a driving factor in the induction of apoptosis, with mitochondria-derived ROS (mtROS) generated by hydrophobic bile acids being implicated as a primary mechanism \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Studies have indicated that hydrophobic bile acids induce a mitochondrial permeability transition, causing the collapse of the mitochondrial membrane potential\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, rupture of the outer mitochondrial membrane, and generation of ROS\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Increased ROS levels are suggested to affect downstream mitochondrial proteases\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, BAX translocation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and the release of cytochrome c\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, thereby inducing apoptosis. However, disruption of the mitochondrial membrane potential by bile acids has also been implicated in mechanisms leading to necrosis\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, suggesting that bile acid-induced cytotoxicity encompasses multiple forms of cell death beyond apoptosis. In this study, we demonstrated that iron sensitizes liver tissues to ferroptosis under hydrophobic bile acid exposure in an ex vivo culture system. mtROS is proposed to react with iron through Fenton reaction, resulting in hydroxyl radical production and lipid peroxidation \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Therefore, bile acids-induced mtROS generation may enhance the susceptibility of cholestatic livers to ferroptosis under iron overload. In the intestinal tract of high fat diet-fed mice, bile acid has been suggested to promote iron uptake through the induction of HIF2a expression\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, it remains unclear whether bile acids enhance iron uptake in the liver under conditions of cholestatic liver disease. Further investigations are required to clarify whether bile acids orchestrate the regulation of both iron metabolism and ROS generation or induce ferroptosis under conditions where iron accumulation occurs as a secondary effect of etiologies, such as inflammation.\u003c/p\u003e \u003cp\u003ePrevious studies have investigated the pathogenic roles of apoptosis and necroptosis in cholestatic liver disease progression. In patients with primary biliary cirrhosis (PBC), upregulation of the FAS ligand, which promotes apoptosis \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and enhanced mixed lineage kinase domain-like protein (MLKL) phosphorylation, which signals necroptosis\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, have been observed. Apoptosis has consistently been considered a pivotal contributor to liver injury and subsequent progression of liver fibrosis in the context of cholestatic liver disease. Indeed, liver injury and fibrosis induced by bile duct ligation (BDL) were attenuated in Fas-deficient lpr mice compared to wild-type mice\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The mechanism underlying apoptosis-mediated disease progression suggests that the phagocytosis of apoptotic bodies by hepatic stellate cells promotes pro-fibrotic activation and fibrogenesis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Several lines of evidence suggest that necroptosis exerts pro-inflammatory and pro-fibrotic effects in various inflammatory conditions, including liver diseases. However, a previous study showed that BDL-induced apoptosis and liver fibrosis were comparable between mice deficient in RIP3, which is a key regulator of necroptosis, and wild-type mice\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Therefore, the role of necroptosis in the progression of cholestatic liver disease remains unclear. In our study, we observed substantial worsening of liver fibrosis in a model of cholestatic liver disease associated with iron overload, suggesting that ferroptosis plays a role in the progression of liver fibrosis. Supporting this observation, the induction of ferroptosis in an iron overload model mouse has been demonstrated to accelerate liver fibrosis\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. This finding implies that, in addition to apoptosis, ferroptosis is a key pathogenic event during the progression of cholestatic liver injury. However, the molecular mechanisms through which ferroptosis promotes liver fibrosis require further investigation. In biliary atresia, inflammation and oxidative stress are prominently observed\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Emerging evidence suggests that ferroptosis exacerbates inflammation through immunogenicity\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. This evidence also implies that ferroptosis is critical in facilitating the progression of chronic pathological states in cholestatic liver disease. Oxidative stress, a hallmark of apoptosis, has been reported to induce the upregulation of heme oxygenase 1 (HO-1), which, in turn, has been suggested to contribute to iron accumulation\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Given that iron accumulation appears to be a secondary effect of oxidative stress, ferroptosis may be considered as a later exacerbating event following apoptosis. However, the exact roles of apoptosis and ferroptosis in disease progression are not fully understood. Apoptosis and ferroptosis are likely interconnected and contribute to the disease progression. Therefore, it is essential to investigate the mechanisms that link these processes in future studies.\u003c/p\u003e \u003cp\u003ePediatric cholestatic liver diseases lack well-defined therapeutic approaches because the factors that contribute to their pathogenesis and progression are unclear. This study highlights the role of iron as an exacerbating factor in pediatric cholestatic liver diseases and suggests that targeting ferroptosis could be a promising strategy for controlling the progression of these diseases. Currently, ursodeoxycholic acid (UDCA), which exerts anti-apoptotic effects, is used as a treatment for cholestatic liver disease, but its used is limited by the delay in the time to liver transplantation or death \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Our findings indicated that beyond apoptosis, iron and ferroptosis could serve as important and potential therapeutic targets. Furthermore, bile acids are widely involved in metabolism \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, immunity\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, and various other processes\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. The relationship between bile acids and ferroptosis identified in this study could have implications in these areas, suggesting its potential application in various diseases.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e (Stock #030532)\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e-Cre (Stock #003574)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e were obtained from Jackson Laboratory. \u003cem\u003eFbxl5\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice were generated as previously described\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These lines were then crossed to generate \u003cem\u003eAlb\u003c/em\u003e-Cre/\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e-Cre/\u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e/\u003cem\u003eFbxl5\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice. All mice were backcrossed to a C57BL/6 background for more than seven generations and maintained under specific pathogen-free conditions. Animals were group-housed in temperature-controlled conditions (22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) with a 12-h light/dark cycle and had ad libitum access to food and water. Unless otherwise noted, experiments were conducted using male mice aged 2\u0026ndash;10 weeks. \u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eYap\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e/\u003cem\u003eFbxl5\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice were used as controls. For the low-iron diet intervention, mother mice were transitioned from a standard diet (CA-1 containing Fe; 30 mg/100 g feed) (CLEA, Tokyo, Japan) to a low-iron diet (AIN-93G containing Fe; 0.32 mg/100 g feed; CLEA) at the time of delivery and allowed ad libitum access to the low-iron diet for 2 weeks. For the NAC treatment, the mothers were switched from standard water (filtered and UV-irradiated tap water) to NAC water (2 g/L, filtered) (Wako, #1705131), which was provided ad libitum for 4 weeks. All mouse experiments were approved by the Animal Ethics Committee of Kumamoto University and the Institute of Science, Tokyo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis of mouse liver\u003c/h2\u003e \u003cp\u003eTo prepare paraffin sections, liver tissues were harvested and fixed immediately in 10% neutral-buffered formalin (Wako, 062-01661) at room temperature overnight. After fixation, liver sections were dehydrated, embedded in paraffin, and cut into 5 \u0026micro;m sections using a microtome (Leica RM2125). The sections were deparaffinized and rehydrated before staining. For Sirius red staining, paraffin sections were stained with Sirius red solution [1% Sirius red (Muto Pure Chemicals, #3306-1): Ven Gieson Solution P (Wako, #224\u0026ndash;01405)\u0026thinsp;=\u0026thinsp;1:30] for 1 h, followed by washing with 0.01 N HCl (Wako, #080-01066) for 1 min. For hematoxylin-eosin (HE) staining, paraffin sections were stained with hematoxylin (Sakura Finetek Japan, #9131-2)and eosin (Sakura Finetek Japan, #9135). For DAB-enhanced Perls staining, paraffin sections were incubated with buffer containing 2% potassium ferrocyanide (II) trihydrate (Wako, 167\u0026ndash;03722) and 0.4% HCl (Wako, #080-01066) for 30 min, followed by development with DAB using a Peroxidase Stain DAB kit (Nacalai Tesque, #23985-50). The sections were dehydrated in xylene after each staining, and coverslips were applied using Mount-Quick (DAIDO SANGYO, #DM-01). Sections were imaged and analyzed using a KEYENCE BZ-X800 all-in-one microscope (KEYENCE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunostaining\u003c/h2\u003e \u003cp\u003eParaffin sections (5 \u0026micro;m) were deparaffinized and rehydrated. Sections were subjected to antigen retrieval by heating in citrate buffer pH 6 [10 mM trisodium citrate dihydrate (Wako, #191\u0026ndash;01785) solution and 10 mM citric acid monohydrate (Wako, #038-03505)] in a microwave (600 W) for 10 min. For immunohistochemistry, sections were subjected to 0.3% hydrogen peroxide (Wako, #081-04215) in methanol for 30 min. After two times washing with dH2O, sections were permeabilized with 0.2% Triton X-100 (polyoxyethylene (10) octylphenyl ether, Wako, #168-11805) in phosphate-buffered saline (PBS) for 10 min. Sections were then washed three times with PBS, followed by blocking with 5% goat serum (Sigma-Aldrich, #G9023-10ML) for 60 min. Sections were washed with PBS and then incubated with a primary antibody in PBS containing 1% bovine serum albumin (BSA) (Wako, #018-15154) at 4\u0026deg;C overnight. After incubation, the sections were washed three times with PBS, followed by sensitization with the VECTASTAIN Elite ABC-HRP kit and peroxidase (Rabbit IgG) (Vector Laboratories, #PK6101) for 30 min. After washing three times, the color was developed using a Peroxidase Stain DAB kit (Nacalai Tesque, #23985-50). Sections were counterstained with hematoxylin and dehydrated with xylene, and coverslips were applied using Mount-Quick (DAIDO SANGYO, #DM-01). For immunofluorescence staining, the sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate buffer, as described above. Thereafter, sections were permeabilized with 0.2% Triton X-100 in PBS for 10 min, followed by blocking with 5% goat serum for 60 min. Sections were then washed and incubated with primary antibody in PBS containing 1% BSA, 0.2% Triton-100 at 4\u0026deg;C overnight. Following three washes, sections were incubated with secondary antibody and DAPI (DOJINDO, #D523) in PBS containing 0.2% Triton X-100 for 60 min at room temperature in the dark. The sections were washed three times, mounted, and coverslipped using Fluoromount (COSMO BIO, K024). The images were acquired using a KEYENCE BZ-X800 all-in-one microscope (KEYENCE). The following antibodies were used: cytokeratin 19 (1:2000 dilution) (Abcam #ab133496), 4-hydroxyneonal (4-HNE) (1:1000 dilution) (Abcam #ab46545), and Alexa Fluor 546-labeled anti-rabbit IgG secondary antibody (1:500 dilution) (Invitrogen #A11030).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTotal non-heme iron measurement\u003c/h2\u003e \u003cp\u003eFrozen liver tissues were thawed to room temperature and washed three times (30 min/each) with PBS, followed by incubation at 45\u0026deg;C for 48 h. The dried tissues were finely crushed and added to an acidic solution [0.6 mM trichloroacetic acid (Nacalai Tesque, #34605-24), 14% HCl (Wako, #080-01066)] and incubated at 65\u0026deg;C for 48 h. Samples were centrifuged at 1,500 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min. The resulting supernatant was mixed with chromogen solution [0.01% bathophenanthroline disulfonic acid disodium salt (Wako, 348\u0026thinsp;\u0026minus;\u0026thinsp;00191), 0.1% mercaptoacetic acid (Nacalai Tesque, #33711-12), and 3.5 M sodium acetate (Wako, #198-15965)], followed by incubation at room temperature for 10 min. The absorbance of each sample was then measured at 535 nm. A dilution series of iron standard solutions (Nacalai Tesque, #37514-24) was prepared, and their absorbance was measured similarly to generate a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSerum analysis\u003c/h2\u003e \u003cp\u003eSerum alanine aminotransferase (ALT), ALP, and bilirubin (total, conjugated, and unconjugated) levels were measured using a standard clinical autoanalyzer (SRL, Inc.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePrecision-cut liver slice (PCLS) culture\u003c/h2\u003e \u003cp\u003eThe liver was excised from the mouse, placed in Kreb\u0026rsquo;s buffer [2.0 g/L D-glucose (Wako, #049-31165), 0.288 g/L magnesium sulfate (Nacalai Tesque, #137\u0026ndash;00402), 0.16 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (Wako, #169\u0026ndash;04245), 0.35 g/L KCl (Wako, #163\u0026ndash;03545), 6.9 g/L NaCl (Wako, #195\u0026ndash;01663), 0.282 g/L CaCl\u003csub\u003e2\u003c/sub\u003e (Wako, #163\u0026ndash;03545), and 2.1 g/L NaHCO\u003csub\u003e3\u003c/sub\u003e (Wako, #191\u0026ndash;01305); pH 7.4] and kept on ice until slicing. The liver lobe was isolated and sliced into 200 \u0026micro;m thickness with a vibratome (Neo LinerSlicer M, DOSAKA EM CO., LTD). The sections were then immediately placed in a 12-well plate containing 250 \u0026micro;L of DMEM supplemented 10% fetal bovine serum (FBS) and incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of bile acid toxicity in PCLS\u003c/h2\u003e \u003cp\u003eThe medium of the PCLS was replaced with a deoxycholic acid (DCA) [sodium deoxycholate (Wako, #192\u0026ndash;08312) in dH2O] dissolved in DMEM containing 10% FBS, followed by incubation at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h. In several experiments, ammonium iron (III) citrate (FAC); Sigma-Aldrich, #F 5879-100G), deferoxamine (DFO) (Abcam, #ab120727), and ferrostatin-1(Fer-1) (Cayman, #17729) were added simultaneously. After incubation, PCLSs were stained with Hoechst33342 (1 mg/mL) (Sigma-Aldrich, #B2261-25G) and PI (1:1000) (DOJINDO, #341\u0026ndash;07881) for 30 min in 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Following a PBS wash, PCLSs were fixed using 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature in the dark. The PCLSs were washed three times, mounted, and coverslipped with Fluoromount. Images were captured using a confocal microscope (FV3000; Evident). Quantification of PI and Hoechst staining was performed using Fiji/ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eInk injection for bile duct visualization\u003c/h2\u003e \u003cp\u003eMice were euthanized, followed by a laparotomy and clipping of the common bile duct. Subsequently, a 29G insulin syringe (TERUMO, #SS-10M2913A) was inserted into the gallbladder, and pigment-based ink (KIWAGURO, SAILOR) was gradually infused to stain the bile ducts retrogradely. After staining, the livers were incubated in 10%, 40%, 80% ethanol in PBS on a gentle rocker at 4\u0026deg;C, each for 1 h, followed by overnight dehydration in 100% ethanol. Following dehydration, BABB solution [benzyl alcohol (Wako, #024-01286):benzyl benzoate (Wako, #025-01336)\u0026thinsp;=\u0026thinsp;1:2] was used to clear the liver by rocking it at 4\u0026deg;C until clearing. Images were captured using a stereoscopic microscope (M165FC; Leica).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblot analysis\u003c/h2\u003e \u003cp\u003eLiver tissues were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100) with protease inhibitors [10 \u0026micro;M Leupeptin (Wako, #336-40413), 1 \u0026micro;M Pepstatin A (Wako, #330-43973) and 1 mM PMSF (Wako, #160-12183)] on ice. After homogenization, samples were kept on ice for 30 min, followed by centrifugation at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min at 4\u0026deg;C. The resulting supernatant was used for analysis. Protein volume was measured by TaKaRa Bradford Protein Assay Kit (Takara, #T9310A), and equal amounts of protein were separated using an 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by transfer onto PVDF membrane (Millipore, #IPVH00010). The membrane was blocked with 5% skim milk (Wako, #190-12865) for 30 min at room temperature, and immunoblotted with primary antibodies in Tris-buffered saline (TBS) containing 0.1% Tween20 and 0.5% skim milk at 4\u0026deg;C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 60 min. Proteins were developed using a chemiluminescent HRP substrate (Millipore, WBKLS0500), and images were captured using a ChemiDoc Touch imaging system (Bio-Rad Laboratories, #17001401JA). The following antibodies were used in the experiments: HRP-conjugated anti-YAP (1:2000 dilution) (Cell Signaling Technology, #15208), anti-IRP2 (1:2000 dilution) (Millipore, #MABS2020-100UG), HSP90 (1:2000) (BD Transduction, #610418), and HRP-conjugated anti-mouse IgG (Cell Signaling Technology, #7026) were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eQuantitative results are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. All statistical analyses were performed using GraphPad Prism 10 software (GraphPad Software, Inc.) according to the methods described in the figure legends. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eOTHER NOTES\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Center for Animal Resources and Development, Kumamoto University, for their support in animal experiments; the Core Laboratory for Medical Research and Education, Kumamoto University School of Medicine, for their technical support; and Hiroyuki Oshiumi for discussions.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Y.O, Y.K, and T.Moroishi; Methodology: Y.O, Y.K, A.M, A.N, T.Matsumoto, K.I.N and T.Moroishi; Investigation: Y.O, Y.K, A.M, M.F.S, and T.Moroishi; Writing of original draft: Y.O, Y.K, and T.Moroishi; Funding acquisition: Y.K, T,M; Supervision: T.Moroishi. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study design was approved by the Animal Committee of Kumamoto University (A2023-031) and Institute of Science Tokyo (A2024-166C).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grants (23K19098, 24H00864, and 24H00865 to TM; 22K15396 and 24K10094 to Y.K), the Japan Science and Technology Agency (JST) FOREST Program (JPMJFR226J to T.M), JST SPRING (JPMJSP2127 to Y.O), and the Kato Memorial Bioscience Foundation (to T.M).\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSutton, H., Karpen, S. J. \u0026amp; Kamath, B. M. 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Despite their clinical importance, the mechanisms driving disease progression remain poorly understood. Here, we report that hepatic iron accumulation is a pathological feature associated with congenital cholestatic liver disease in mice with a liver-specific deletion of \u003cem\u003eYap\u003c/em\u003e, a gene critical for bile duct development. We demonstrated that further hepatic iron overload induced by liver-specific deletion of \u003cem\u003eFbxl5\u003c/em\u003e, a key regulator of cellular iron homeostasis, exacerbated cholestatic liver injury and fibrosis in \u003cem\u003eYap\u003c/em\u003e-deficient mice. Mechanistically, iron overload enhanced the susceptibility to bile acid-induced cytotoxicity via ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation. This ferroptotic process was confirmed by the suppression of bile acid-induced cell death through iron chelation and lipid peroxide scavenging in \u003cem\u003eex vivo\u003c/em\u003e liver slice cultures. Furthermore, both dietary iron restriction and antioxidant treatment mitigated liver injury \u003cem\u003ein vivo\u003c/em\u003e. These findings identify iron accumulation as a key driver of disease progression and highlight iron metabolism and ferroptosis as potential therapeutic targets in congenital cholestatic liver disease.\u003c/p\u003e","manuscriptTitle":"Iron exacerbates congenital cholestatic liver injury via bile acid-induced ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-23 15:01:02","doi":"10.21203/rs.3.rs-6671814/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48cce247-d20b-49c7-9fef-8024c8c5c0bd","owner":[],"postedDate":"May 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48838434,"name":"Biological sciences/Cell biology/Cell death"},{"id":48838435,"name":"Biological sciences/Biochemistry/Metals/Iron"}],"tags":[],"updatedAt":"2025-05-23T15:01:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-23 15:01:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6671814","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6671814","identity":"rs-6671814","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}