Loss of Cdkn1a protects against MASLD alone or with alcohol intake by preserving lipid homeostasis

Loss of Cdkn1a protects against MASLD alone or with alcohol intake by preserving lipid homeostasis Authors Arantza Lamas-Paz, Alejandro Hionides-Gutiérrez, Feifei Guo, ., Pere Puigserver, Yulia A. Nevzorova, Francisco Javier Cubero Correspondence [email protected] (F.J. Cubero). Graphical abstract Cdkn1a AMPK Cdkn1a-/- AKT activation FAO FFA uptake de novo lipogenesis SIRT3 DUAL e c n e c s e n e s C57/B6J Fibrosis Oxidative stress Metabolic syndrome Steatosis EXPERIMENTAL WD Palbociclib CLINICAL CDKN1A e c n e c s e n e s Metabolic syndrome Steatosis Fibrosis Inflammation Inflammation MASLD Cirrhosis Role of Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A) in steatotic liver disease Highlights: CDKN1A contributes to the stability of cell cycle arrest after the induction of senescence. Liver senescence is responsible for the metabolic shifts causing fat accumulation and hepatic inflammation in patients with SLD. Loss of Cdkn1a has a protective effect in preclinical metabolically induced SLD, with and without alcohol consumption. CDKN1A expression correlates with NAFLD activity score and advanced fibrosis in patients with MASLD. Impact and implications: Expression of p21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD), but the molecular mechanisms remain elusive. Interestingly, in this study we found that Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis, via the AMPK-SIRT3 axis. Translationally, Cdkn1a expression was found to be directly correlated with increased severity of NAFLD Activity Score (NAS) and fibrosis in SLD patients, and therefore, CDKN1A might be used potential theragnostic target for the treatment of metabolically induced SLD, with and without alcohol consumption. https://doi.org/10.1016/j.jhepr.2024.101230 © 2024 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). JHEP Reports, 2024, -,1–15 Research article 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 Loss of Cdkn1a protects against MASLD alone or with alcohol intake by preserving lipid homeostasis Arantza Lamas-Paz 1,2,† ,Alejandro Hionides-Gutiérrez 1,† ,Feifei Guo 1,3,† , Gonzalo Jorquera 4,5 , Laura Morán-Blanco 1 , Raquel Benedé-Ubieto 1 , Mariana Mesquita 1,6 , Olga Estévez-Vázquez 1 , Kang Zheng 1,7 , Marina Mazariegos 1 , Elena Vázquez-Ogando 8,9,10 , Elena Blázquez-López 8,9,10 , Iris Asensio 8,9,10 , Beste Mutlu 11,12 , Beatriz Gomez-Santos 13,14 , María Isabel Peligros 15 , Javier Vaquero 8,9,10 , Rafael Bañares 8,9,10 , Teresa C. Delgado 16 , María Luz Martínez-Chantar 10,16 , Eduardo Martínez-Naves 1,2 , Carlos Sanz-García 1 , Mohamed Ramadan Mohamed 17 , Sofía Tesolato 18,19 , Pilar Iniesta 18,19 , Rocío Gallego-Durán 10,20 , Douglas Maya-Miles 10,20 , Javier Ampuero 10,20 , Manuel Romero-Gómez 10,20 , Ana Martínez-Alcocer 10,21 , David Sanfeliu-Redondo 10,21 , Anabel Fernández-Iglesias 10,21 , Jordi Gracia-Sancho 10,21,22 , Mar Coll 10,23 , Isabel Graupera 10,23,24 , Pere Ginès 10,23,24 , Andrea Ciudin 25,26 , Jesús Rivera-Esteban 27,28 , Juan M. Pericàs 10,27 , Matías A. Ávila 10,29,30 , Maria Dolores Frutos 31 , Carlos Manuel Martínez-Cáceres 32 , Bruno Ramos-Molina 33 , Patricia Aspichueta 10,13,14 , Pere Puigserver 11,12,‡ , Yulia A. Nevzorova 1,9,10,‡ , Francisco Javier Cubero 1,9,10, * ,‡ JHEP Reports 2025. vol. 7 j1–15 Background & Aims: Expression of P21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD); however, the underlying mechanisms remain unknown. In the present study, we investigated the function of CDKN1A in SLD. Methods: CDKN1A expression levels were evaluated in different patient cohorts with SLD, fibrosis, and advanced chronic liver disease (ACLD). Cdkn1a -/- and Cdkn1a +/+ mice were fed with either a Western diet (WD), a Lieber-DeCarli (LdC) diet plus multiple EtOH (ethanol) binges, or a DuAL diet (metabolic dysfunction-associated fatty liver disease and alcohol-related liver). Primary hepatocytes were isolated and functional assays performed. Results: A significant increase in CDKN1A expression was observed in patients with steatohepatitis and fibrosis (with a positive correlation with both NAFLD Activity Score and fibrosis staging scores), cirrhosis and ACLD. Cdkn1a +/+ mice, fed a DuAL diet exhibited liver injury and cell death increased reactive oxygen species (ROS), and markers of senescence (cH2AX, b-GAL, Cdkn1a/ p53) contributing to steatosis and inflammation. In contrast, Cdkn1a -/- mutant mice showed a significant decrease in senescenceassociated markers as well as in markers of liver injury, hepatic steatosis and an increase in fatty acid oxidation and reduction in free fatty acid uptake as well as de novo lipogenesis. Mechanistically, activation of the AMPK-SIRT3 was observed in Cdkn1adeleted animals. Conclusions: Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis via the AMPK-SIRT3 axis. CDKN1A expression was found to be directly correlated with increased severity of NAFLD Activity Score and fibrosis in patients with SLD. CDKN1A could be a potential theragnostic target for the treatment of metabolic dysregulation in patients with SLD, with and without alcohol consumption. © 2024 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction Cyclin-dependent kinase inhibitor 1A (CDKN1A) is a member of the cyclin-dependent kinases inhibitors (CDKN) of the Cip/Kip family 1 that arrests cells by affecting the activity of cyclin D-, E-, and A-dependent kinases, which regulate progression through the G1 phase of the cell cycle and inhibition of DNA synthesis. 2 CDKN1A also inhibits proliferation competing with proliferating cell nuclear antigen (PCNA) 3 or indirectly at the transcriptional level. 4 CDKN1A contributes to the stability of cell cycle arrest long after the induction of senescence. 5 Microarray-based studies suggested that CDKN1A expression positively correlates with both the suppression of genes involved in cell cycle progression, and the induction of senescence genes. 5 Cellular senescence develops in response to cellular injury, leading not only to cell cycle arrest, but also to alterations of the cellular phenotype and metabolic functions. 6 A plethora of evidence suggested that liver senescence, and particularly hepatocytic senescence, is responsible for the metabolic shifts causing fat accumulation and liver inflammation in patients with steatotic liver disease (SLD), the main overarching cause of chronic liver disease (CLD). 7 * Corresponding author. Address: Department of Immunology, Ophthalmology and ENT, Complutense University School of Medicine, c/Doctor Severo Ochoa, 9, 28040, Madrid, Spain. Tel.: +34 91394 1385. E-mail address: [email protected] (F.J. Cubero). † Authors contributed equally as first authors. ‡ Authors contributed equally as senior authors. https://doi.org/10.1016/j.jhepr.2024.101230 JHEP Reports, --- 2025. vol. 7 j101230 Research article CLD represents a major health problem, representing the 10th cause of death worldwide, with 2 million individuals dying of liver disease each year. 8 CLD is defined as the progressive deterioration of liver functions with a continuous process of inflammation, destruction, and regeneration of liver parenchyma, which leads to fibrosis and cirrhosis. 9 The spectrum of etiologies is broad for CLD, including SLD with or without potentially harmful alcohol intake, the latter known as MetALD. 10 Metabolic dysfunction-associated SLD (MASLD), its more rapidly growing form, ranges from steatosis to steatohepatitis and cirrhosis. Moreover, metabolic dysfunction can synergize with harmful alcohol consumption (MetALD), which can induce SLD (alcohol-related liver disease [ALD]). Both MASLD and MetALD are hepatic insults that range from simple steatosis to advanced CLD, including cirrhosis and hepatocellular carcinoma (HCC). 11 Several studies showed an association between CDKN1A expression and the progression of CLD. 12–16 Increased CDKN1A expression is evidenced in animal models of obesity 14,17 and in patients with SLD. 15 Therefore, we sought to investigate whether the development of SLD and its progression to steatohepatitis, commonly accompanied by several pathophysiological events including metabolic dysregulation and inflammatory phenomena occurring within the liver, may derive from CDKN1A-derived induction of cellular senescence, triggering metabolic alterations. Moreover, we explored the pharmacological inhibition of CDKN1A in preclinical SLD using palbociclib. Altogether, our data strongly support the notion that CDKN1A has a protective effect against preclinical metabolically induced SLD, with and without alcohol consumption, and might be an optimal theragnostic tool for the progression of CLD. Materials and methods Patient cohorts This multicentric study was composed of several cohorts. Cohort #1 is a metanalysis (Table S1) that integrated data from three different studies, mostly patients with MASLD, with different stages of fibrosis (F0–F4), in which expression of CDKN1A was analyzed using RNAseq. All data were integrated into a single analysis in which each study was treated as a batch. Cohort #2 consisted of 91 consecutive and prospective individuals recruited for a bariatric intervention in the Virgen de la Arrixaca University Hospital (Murcia, Spain), with suspicion of MASLD. 18 The study was approved by the Ethics and Clinical Research Committees of the Virgen de la Arrixaca University Hospital (ref. number 2020-2-4-HCUVA). A recently characterized cohort (cohort #3) (22 patients) 19 encompassing the whole spectrum of CLD stages to any cause of SLD, ranging from patients with early fibrosis to compensated cirrhosis (CC) was also used. F1/F2/F3 fibrosis, named early-CLD to CC was also used. The protocol of the study was approved by the Institutional Review Board of the Hospital Clinic of Barcelona (code: 2012/7977). Cohort #4 consisted of 13 patients from a previously published study 20 with advanced CLD (decompensated cirrhosis) that underwent liver transplantation and control patients with no suspected CLD, who underwent liver resection as a result of colorectal carcinoma metastasis. The study’sfirst stage, in which the miRNA signature was identified, was conducted at the August Pi i Sunyer Biomedical Research Institute-Hospital Clinic of Barcelona, and its protocol was approved by this center’s Ethics Committee (HCB/2018/0028). The protocol for subsequent stages received approval from the Ethics Committee for Clinical Research of the Hospital Universitario Ramon y Cajal (institutional review board number 362/19; approval date April 1, 2019). All included individuals provided written informed consent authorizing the storage and research use of their biological samples. Animal experimentation Eight-to 13-week-old male Cdkn1a -/- knockout mice (B6;129S2 Cdkn1atm1Tyj/J, The Jackson Laboratory, Bar Harbor, ME, USA) and Cdkn1a +/+ mice (B6;129SF2/J, The Jackson Laboratory) were maintained in the animal Facility of the Faculty of Biology at Complutense University of Madrid (UCM), in a temperatureand humidity-controlled room with a 12-h light/ dark cycle and allowed food and water ad libitum, according to the guidelines of the Federation for Laboratory Animal Science Associations (FELASA). All animal procedures were carried out according to Spanish legal requirements and animal protection law and approved by the authority of environment conservation and consumer protection of the Regional Government of Madrid (PROEX-397.2/21). Cdkn1a -/- and Cdkn1a +/+ mice were fed with a DuAL diet (metabolic dysfunction-associated fatty liver disease and alcohol-related liver; n = 6–10) consisting of Western diet (WD) (D18121807, Research Diets, Inc., New Brunswick, NJ, USA) and 10% vol/vol absolute ethanol (EtOH) in drinking sweetened water (6.75% D-glucose, Merck, Madrid, Spain) for 18 weeks 21 ; a WD (D18121807, Research Diets, Inc.) (n = 8–12) for 14 weeks and an EtOH diet (n = 7–13) where mice were allowed free access to 5.07% w/v (6.4% v/v) EtOH Lieber-DeCarli (LdC) liquid diet (F1258SP, Bio-Serv, Madrid, Spain) for 8 weeks plus a 30% EtOH gavage (6 g/kg body weight [BW]) every 2 weeks. 22 C57BL/6 WT mice were also fed with DuAL diet for 10 weeks, plus an administration of 100 mg/kg of palbociclib (MedChemExpress, Madrid, Spain) via oral gavage twice a week. Controls were fed with normal chow diet (Altomin, Lage, Germany) and filtered tap water or LdC control diet, where the remainder of the energy is provided as carbohydrates, which in the EtOH formula was isocalorically replaced by EtOH. At the end of each experiment, all animals were fasted overnight for 12 h and sacrificed by an overdose of isoflurane (Solvet, Segovia, Spain) and liver and blood were collected. Statistical analysis Data are expressed as mean ± SD. GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA) was used to perform statistical analysis. Data are expressed as mean ± SD. For each experiment, statistical details can be found in the figure legends, including statistical tests and sample sizes. To evaluate the statistical differences between Cdkn1a -/- and Cdkn1a +/+ mice and siCtrl and siCdkn1a primary hepatocytes in functional assays ANOVA one-way Tukey post hoc test was used. Additionally, statistical significance between siCtrl and siCdkn1a primary hepatocytes in quantitative PCR (qPCR) assays was determined using Student ttest. In general, a JHEP Reports, --- 2025. vol. 7 j101230 2 Role of Cdkn1a in MASLD value of p<0.05 was considered significant. Pearson correlation was utilized to assess the relationship between gene expressions. Results Expression of CDKN1A and SASP in patients with MASLD/ MASH and fibrosis Significantly increased gene counts for CDKN1A were observed in a RNAseq dataset of patients with diagnosed MASLD with NAFLD Activity Score (NAS) >4(Fig. 1A, cohort #1). Thus, we next analyzed CDKN1A protein expression in liver extracts of patients with MASLD. Interestingly, we observed progressive overexpression of CDKN1A in patients with high NAS score (Fig. S1A, cohort #2). Considering that hepatic fibrosis is an important factor affecting the prognosis of liver diseases, we next evaluated whether CDKN1A was also significantly upregulated in cohorts of patients with fibrosis. CDKN1A gene counts significantly increased from F1 to F4 (Fig. S1B, cohort #1), and, especially, between F0–F2 and F3–F4 (Fig. 1B, cohort #1). Liver fibrosis involves cell cycle re-entry and proliferation of parenchymal and non-parenchymal liver cells controlled by cyclins and associated cyclin-dependent kinases (CDKs). Hence, all cyclins and CDKs tested in cohort #1 were significantly downregulated in F0–F2 compared with F3–F4 (Fig. S1C), indicating the importance of cell cycle in hepatic fibrogenesis. Of note, the area under the receiver operating characteristic curve (AUROC) when assessing CDKN1A was 0.81 (95% CI 0.72–0.89) for fibrosis prediction (Fig. 1C). To validate the utility of CDKN1A in patients with MASLD patients, we examined the correlation of CDKN1A with the NAS and the fibrosis score. CDKN1A positively correlated with NAS (Fig. 1D, Fig. S1D, cohort #1) and the fibrosis score (Fig. 1E, Fig. S1E, cohort #1), highlighting the prognostic value of this gene in MASLD and fibrosis. Moreover, increased mRNA expression of CDKN1A was observed in patients with early CLD with F2/F3 fibrosis score J 0 10 20 30 40 CDKN1A-positive cells (HPF, 20x) * Control MASH I MASH CDKN1A H -10 -5 0 5 10 0 2 4 6 8MASLD validation (N = 225) R2 = 0.28 p = <0.0001 CDKN1A expression (normalized counts) IL-8 expression (normalized counts) 0 1 2 3 CDKN1A (fold change) SA ACLD *** G SA F2-F3 CC DC 6 7 8 9 10 CDKN1A (fold change) *** FE 01234 R2 = 0.25 p <0.0001 0 2 4 6 8 10 Fibrosis stage MASL validation (RNAseq n = 225) CDKN1A expression (normalized counts) D 02468 0 2 4 6 8 10 NAS score MASL validation (RNAseq n = 187) CDKN1A expression (normalized counts) R2 = 0.12 p <0.0001 CCDKN1A: 0.81 [0.72-0.89] 1-Specificity 1.00.80.60.40.20.0 Sensitivity 1.0 0.8 0.6 0.4 0.2 0.0 B 0 5 10 MASLD cohort (RNAseq n = 225) **** F0-F2 F3-F4 CDKN1A expression (normalized counts) A 0 2 4 6 8 10 MASLD validation (n = 187) * MASLD MASH CDKN1A expression (normalized counts) Fig. 1. CDKN1A expression is characteristic of patients with SLD and fibrosis. (A) Cohort #1. CDKN1A mRNA relative expression to GAPDH in livers of patients with MASLD and MASH, identified with a NAS score > −5 (n = 187). (B) Cohort #1. CDKN1A mRNA relative expression to GAPDH in livers of patients with MASLD with a fibrosis score between F0–F2 and F3–F4 (n = 225). (C) Cohort #1. AUROC when assessing CDKN1A for fibrosis prediction. AUROC of 0.81 (95% CI 0.72–0.89) for fibrosis prediction. (D) Cohort #1. Correlation between CDKN1A mRNA relative expression to GAPDH in livers of patients with MASLD with a NAS score >4 (n = 187). (E) Cohort #1. Correlation between CDKN1A mRNA relative expression to GAPDH in livers of patients with MASLD with a fibrosis score F0–F2 and F3–F4 (n = 225). (F) Cohort #3. CDKN1A mRNA relative expression to GAPDH in livers of non-affected patients and patients with a fibrosis score between F2 and F4 and compensated cirrhosis (CC) and decompensated cirrhosis (DC) (n = 22). (G) Cohort #4. CDKN1A mRNA relative expression to GAPDH in livers of patients with ACLD and nonaffected patients (n = 13). (H) Cohort #1. Correlation between IL-8 mRNA relative expression to GAPDH in livers of patients with MASLD. (I) Immunostaining for CDKN1A was tested in paraffin sections of patients with a diagnosis of MASH with the presence of steatosis and inflammation. Microphotographs were taken at 300 l m. (J) Quantification of positive cells was performed and graphed, 200 hepatocytes per field were counted. Student ttest was performed (*p<0.05; ****p<0.0001). ACLD, advanced chronic liver disease; AUROC, area under the receiver operating characteristic curve; CDKN1A, cyclin-dependent kinase inhibitor 1A; GADPH, glyceraldehyde-3-phosphate dehydrogenase; MASLD, metabolic dysfunction-associated steatotic liver disease; MASH, metabolic-associated steatohepatitis; NAS, NAFLD Activity Score; SLD, steatotic liver disease. JHEP Reports, --- 2025. vol. 7 j101230 3 Research article (Fig. 1F, cohort #3), CC and decompensated cirrhosis (DC), and with advanced CLD (Fig. 1G, cohort #4). Inflammation is a significant component of MASLD and is closely linked to the senescence-associated secretory phenotype (SASP). Therefore, we analyzed the correlation between the levels of proinflammatory cytokines in patients with MASLD. Interestingly, we found a significant correlation of IL8 (Fig. 1H, cohort #1), and IL6ST with patients with MASLD (Fig. S1F, cohort #1). Finally, to understand hepatocytic or nonparenchymal cells (NPC)-associated expression of CDKN1A/ P21, we performed immunohistochemical (IHC) staining in paraffin sections of patients diagnosed with MASLD/metabolicassociated steatohepatitis (MASH). Interestingly, CDKN1A expression was restricted to hepatocyte nuclei and characteristic of patients with MASLD/MASH compared with nonaffected tissue (normal) (Fig. 1I,J, Fig. S1G, cohort #2). Loss of Cdkn1a protects mice from liver injury after a DuAL diet To understand the theragnostic value of CDKN1A in the progression of liver disease, we first analyzed the mRNA expression of Cdkn1a in isolated liver cells. Interestingly, Cdkn1a expression was highly expressed in hepatocytes, but also in NPCs-hepatic stellate cells (HSCs) and Kupffer cells (KCs) (Fig. S2A). We subsequently evaluated Cdkn1a expression in preclinical models of MASLD/MetALD. Cdkn1a mRNA expression was significantly upregulated in wildtype mice after DuAL and WD diet compared with chow-fed animals, whereas no differences were observed in the EtOH-fed group (Fig. S2B). At the protein level, overexpression of CDKN1A was characteristic of mice fed a DuAL diet (Fig. S2C). These results indicated that CDKN1A might play an important role in preclinical MASLD with alcohol intake. Therefore, we subsequently focused on examining the role of CDKN1A in the DuAL model, which mimics the clinical features of MASLD and chronic moderate harmful alcohol consumption during 18 weeks of treatment. Cdkn1a +/+ animals fed a DuAL diet exhibited enlarged livers and significantly elevated liver weight (LW) and LW/body weight (BW) ratio, albeit no differences in BW (Fig. 2A, Fig. S3A–C). However, this increase was significantly reduced in Cdkn1a -/- compared with Cdkn1a +/ + mice (Fig. 2A, Fig. S3A–C). Concomitantly, serum markers of liver damage including alanine aminotransferase (ALT) (Fig. 2B), aspartate aminotransferase (AST) and lactate dehydrogenase (Figs. S3D and E) were significantly decreased in Cdkn1a -/- compared with Cdkn1a +/+ mice fed a DuAL diet. Histopathologically, Cdkn1a +/+ livers displayed hepatocyte enlargement, ballooning, steatosis, and cell death (Fig. 2C, Fig. S3F). DuAL-fed mice exhibited the first signs of fibrosis as soon as 10 weeks of injury with remarkable escalation of fibrogenesis at 23 weeks. 21 These findings were additionally confirmed in Cdkn1a +/+ mice, fed a DuAL diet for 18 weeks, as observed by significantly increased a Sma and CollagenIA1 mRNA expression, Sirius Red and Vimentin IHC stainings (Fig. 2D–F, Fig. S3G,H,Fig. S4A,B). However, these parameters were significantly decreased in Cdkn1a -/- animals (Fig. 2D–F, Fig. S3G,H,Fig. S4A,B). As liver fibrosis is a common feature of chronic liver injury and is initiated by cell death inside the liver, we next studied the levels of proteins related to liver cell death. Interestingly, cleaved caspase-3 (CC3) and -8 (CC8), pRIPK1/3, and pMLKL were significantly overexpressed in Cdkn1a +/+ , compared with Cdkn1a -/- mice fed a DuAL diet (Fig. 2G). Consistently, cell death, measured by terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, was also significantly higher in Cdkn1a +/+ animals, whereas it was significantly decreased in Cdkn1a -/- mice fed a DuAL diet (Fig. 2H). Because of the role of CDKN1A in the progression of cell cycle in the G1 phase as an inhibitor, 12 we evaluated the mRNA transcripts of cell cycle mediators controlling the late G1-and S-phase progression. Therefore, we measured the number of transcripts of cyclins including CcnA2, D1, and E1 (Fig. 2I–K). Interestingly, the levels of CcnA2,D1 transcripts were significantly decreased in Cdkn1a -/- animals, whereas no differences were found in CcnE1 mRNA expression between Cdkn1a +/+ and Cdkn1a -/- (Fig. 2I–K). Furthermore, no statistical significance was observed in Ki67-positive cells in Cdkn1a -/- , compared with Cdkn1a +/+ animals fed a DuAL diet (Figs. S4C and D). Deletion of Cdkn1a protects against preclinical SLD As the DuAL preclinical model represents the combined synergistic effect of both a WD and harmful alcohol intake, we next examined the role of CDKN1A in each of the factors alone. Therefore, WD treatment was performed over 14 weeks in Cdkn1a +/+ and Cdkn1a -/- mice (Fig. 3). Interestingly, a significant decrease in the LW/BW ratio (Fig. 3A, Fig. S5A), as well as in serum markers of liver damage –ALT (Fig. 3B) and AST (Fig. S5B) were observed in Cdkn1a -/- mice fed a WD. The histopathological study of liver sections by H&E showed macrosteatosis mainly surrounding the portal spaces and predominant microsteatosis located in the perivenular areas of Cdkn1a +/+ animals, fed a WD (Fig. 3C, Fig. S5C). However, liver architecture was preserved in Cdkn1a -/- mice. Moderately enhanced chicken wire fibrosis appeared along with sinusoidal and periportal fibrillar collagen deposition in both Cdkn1a +/+ and Cdkn1a -/- mice fed a WD diet (Fig. 3D,F, Fig. S5D). Apart from this mild fibrosis, the WD triggered cell death in the hepatic parenchyma, analyzed as TUNEL-positive cells in Cdkn1a +/+ . Interestingly, mice with deletion of Cdkn1a displayed significantly decreased TUNEL positivity (Fig. 3E,G, Fig. S5E), albeit no differences in cell proliferation (Fig. 5F,G). Furthermore, we assessed insulin resistance using the glucose tolerance test (GTT) in mice after WD feeding. GTT results and basal glucose levels measured in blood in Cdkn1a +/ + animals showed a delay in glucose uptake after WD; however, not significant compared to Cdkn1a -/- mice (Fig. S6A). The area under the curve of the GTT test exhibited a statistically significance disparity only between the chow and WD-fed Cdkn1a -/- mice. However, no statistical significance was observed in Cdkn1a +/+ mice when compared with Cdkn1a -/- mice (Fig. S6B). Of note, basal levels of glucose in blood were significantly lower in Cdkn1a -/- mice after a chow diet (Fig. S6C), indicating that metabolism might be dysregulated in this mouse strain. Altogether, these results suggest that Cdkn1a deletion protects against a preclinical model of SLD. JHEP Reports, --- 2025. vol. 7 j101230 4 Role of Cdkn1a in MASLD CDKN1A knockout mice are not protected against murine ALD To further explore the relevance of CDKN1A in preclinical ALD, we performed an acute-on-chronic LdC diet plus multiple EtOH binges, a modification of the National Institute on Alcohol Abuse and Alcoholism model, 22 in Cdkn1a +/+ and Cdkn1a -/- mice. No differences were observed macroscopically in livers or in the LW/BW ratio between Cdkn1a +/+ and Cdkn1a -/- animals (Fig. 3H, Fig. S7A). Accordingly, serum markers of liver damage (ALT, AST) were elevated in both Cdkn1a +/+ ,andCdkn1a -/- fed a LdC, compared with control diet-fed mice (Fig. 3I, Fig. S7B). Alcohol metabolism by CYP2E1 (cytochrome P450) levels showed a similar induction K n.s. * Chow DuAL Chow DuAL 0 1 2 3 4 5 CcnE1 (fold change) Cdkn1a+/+ Cdkn1a-/- ** J### ** Chow DuAL Chow DuAL 0 1 2 3 4 5 CcnD1 (fold change) n.s. I ** ## Chow DuAL Chow DuAL 0 2 4 6 8 CcnA2 (fold change) n.s. Cdkn1a+/+ Cdkn1a-/- H **** ## Chow DuAL Chow DuAL 0.0 0.2 0.4 0.6 0.8 TUNEL positive cells (%) n.s. G Chow DuAL DuAL pRIPK1 74 pRIPK3 57 GAPDH 37 pMLKL 50 CC3 19 17 CC8 18 50 CC8 Chow 1.39 ± 0.30 1.71 ± 0.11 1.01 ± 0.13 0.63 ± 0.10# 0.20 ± 0.06 1,43 ± 0.25** 0.64 ± 0.05 0.47 ± 0.11## 0.11 ± 0.02 0.94 ± 0.14** 0.36 ± 0.03 0.38 ± 0.00## 0.14 ± 0.02 0.68 ± 0.03*** 0.13 ± 0.04 0.05 ± 0.03### 0.68 ± 0.17 0.66 ± 0.03 0.67 ± 0.08 0.18 ± 0.01# kDa Cdkn1a+/+ Cdkn1a-/- F Sirius red DuAL Cdkn1a+/+ Cdkn1a-/- E Chow DuAL Chow DuAL 0.0 0.5 1.0 1.5 2.0 Sirius red area (%) ## * **** Cdkn1a+/+ Cdkn1a-/- D #### ** Chow DuAL Chow DuAL 0 1 2 3 4 α-Sma (fold change) n.s. C H&E DuAL Cdkn1a+/+ Cdkn1a-/- n.s. B Cdkn1a+/+ Cdkn1a-/- # * Chow DuAL Chow DuAL 0 50 100 150 200 250 ALT (U/L) Cdkn1a+/+ Cdkn1a-/- 0 weeks 18 weeks Sacrifice DuAL diet WD 6.75% D-Glucose + 10% EtOH 8 week-old A#### **** Chow DuAL Chow DuAL 0 2 4 6 8 10 LW/BW (%) ** Fig. 2. Liver injury is significantly reduced after a DuAL diet in Cdkn1a -/- mice. Schematic description of a DuAL model performed in Cdkn1a +/+ and Cdkn1a -/- mice. (A) Liver weight to body weight ratio (%). (B) Serum ALT levels. (C) H&E staining in Cdkn1a +/+ and Cdkn1a -/- mice after DuAL diet. Scale bar = 100 l m. (D) a -Sma mRNA relative liver expression to Gapdh. (E) Sirius red staining quantification in Cdkn1a +/+ and Cdkn1a -/- mice after DuAL diet and (F) representative paraffin liver sections stained. Scale bar = 100 l m. (G) Total liver protein (two individuals’samples from each group are shown as representative of the group) were isolated from DuAL diet-fed Cdkn1a +/+ and Cdkn1a -/- mice and analyzed for CC-3, CC-8, pRIPK1, pRIPK3, and pMLKL. Protein expression levels were normalized to the levels of total GAPDH and the ratio was calculated. (H) Quantification of TUNEL-positive cells (%) was done and graphed. (I) CcnA2, (J) CcnD1, and (K) CcnE1 measured in mice of each group relative to Gapdh. Scale bar = 100 l m. n =6–10; one-way ANOVA with post hoc Tukey test was used for the calculations (intragroup: *p<0.05, ****p <0.0001; intergroup: # p<0.05, #### p<0.0001). ALT, alanine aminotransferase; BW, body weight; CC3, cleaved-caspase-3; CC8, cleaved-caspase-8; CcnA2, ciclyn A2; CcnD1, cyclin D1; CcnE1, ciclin E1; Cdkn1a, cyclin-dependent kinase inhibitor 1A; DuAL, metabolic dysfunction-associated fatty liver disease and alcohol-related liver; GADPH, glyceraldehyde-3-phosphate dehydrogenase; LW, liver weight; RIPK, receptor interacting protein kinase; TUNEL, terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling. JHEP Reports, --- 2025. vol. 7 j101230 5 Research article of this enzyme after LdC in both Cdkn1a +/+ and Cdkn1a -/- mice (Fig. S7C). Liver histology analyses showed obvious macro and microsteatosis in the livers of all LdC-fed mice, often accompanied by the presence of inflammatory cells and necrosis (Fig. 3J, Fig. S7D). Next, we measured deposition of extracellular matrix and liver fibrosis as a consequence of LdC feeding, using Sirius Red staining. Both Cdkn1a +/+ and Cdkn1a -/- fed a LdC, displayed increased fibrogenesis, compared with control diet-fed mice (Fig. 3K,M, Fig. S7E,F), albeit no differences between both Nn.s. Chow EtOH Chow EtOH 0.0 0.1 0.2 0.3 0.4 0.5 TUNEL positive-cells (%) Cdkn1a+/+ Cdkn1a-/- ** ** n.s. Chow EtOH Chow EtOH 0.0 0.5 1.0 1.5 2.0 2.5 Sirius red area (%) ** * M L TUNEL Cdkn1a+/+ Cdkn1a-/- K Sirius red Cdkn1a+/+ Cdkn1a-/- H&E EtOH Cdkn1a+/+ Cdkn1a-/- J In.s. Chow EtOH Chow EtOH 0 50 100 150 200 250 ALT (U/L) *** *Cdkn1a+/+ Cdkn1a-/- *n.s. n.s. Chow EtOH Chow EtOH 2.5 3.0 3.5 4.0 4.5 5.5 5.0 LW/BW (%) H G *** 0.0 0.5 1.0 1.5 TUNEL positive-cells (%) Chow Western Chow Western ### n.s. Cdkn1a+/+ Cdkn1a-/- *** 0.0 0.2 0.4 0.6 Sirius red area (%) Chow Western Chow Western n.s.n.s. F E TUNEL Cdkn1a+/+ Cdkn1a-/- D Sirius red Cdkn1a+/+ Cdkn1a-/- H&E Western Cdkn1a+/+ Cdkn1a-/- C B### *** Chow Western Chow Western Cdkn1a+/+ Cdkn1a-/- 0 100 200 300 400 500 ALT (U/L) n.s. A#### **** Chow Western Chow Western 0 2 4 6 8 10 LW/BW (%) n.s. 0 weeks 1 week 3 weeks 9 weeks Lieber-DeCarli EtOH diet (5% EtOH) 5 weeks 7 weeks 30% EtOH Gavage 30% EtOH Gavage 30% EtOH Gavage 0 weeks 14 weeks SacrificeWD diet WD Cdkn1a+/+ Cdkn1a-/- 8 week-old SacrificeLdC diet Adaptation Cdkn1a+/+ Cdkn1a-/- 8 week-old Fig. 3. Deletion of Cdkn1a in a preclinical model of SLD and in murine ALD. (Left panel) Schematic description of a WD model performed in of Cdkn1a +/+ and Cdkn1a -/- mice. (A) LW/BW ratio (%). (B) ALT measurement in serum (n = 7–11). (C) H&E representative images in liver of Cdkn1a +/+ and Cdkn1a -/- mice after WD. Scale bar = 100 l m. (D) Representative liver images stained with Sirius red (E) TUNEL staining was performed in liver cryosections of Cdkn1a +/+ and Cdkn1a -/- mice after WD and (F,G) quantification of positive Sirius Red-stained area and TUNEL-positive cells. Scale bar = 100 l m. (n = 8–12); one-way ANOVA with post hoc Tukey test was used for the calculations (intragroup: ***p<0.001, ****p<0.0001; intergroup ### p<0.001, #### p<0.0001). (Right panel) Schematic description of an LdC model plus multiple binges performed in Cdkn1a +/+ and Cdkn1a -/- mice. (H) LW/BW ratio (%). (I) ALT measurement in serum. (J) H&E staining in Cdkn1a +/+ and Cdkn1a -/- mice after LdC EtOH diet. Scale bar = 100 l m. (I) Sirius Red staining performed in liver of Cdkn1a +/+ and Cdkn1a -/- mice after LdC EtOH diet. Scale bar = 100 l m. (J) TUNEL staining was performed in liver cryosections of liver of Cdkn1a +/+ and Cdkn1a -/- mice after LdC EtOH diet. Scale bar = 100 l m. (M,N) Quantification of Sirius Redpositive area and TUNEL-positive cells was done and graphed, respectively. n = 7–8; one-way ANOVA with post hoc Tukey test was used for the calculations (intragroup: *p<0.05, ****p<0.0001). ALD, alcohol-related liver disease; ALT, alanine aminotransferase; BW, body weight; Cdkn1a, cyclin-dependent kinase inhibitor 1A; EtOH, ethanol; LdC, Lieber-DeCarli; LW, liver weight; SLD, steatotic liver disease; WD, Western diet. JHEP Reports, --- 2025. vol. 7 j101230 6 Role of Cdkn1a in MASLD strains were found. Concomitantly, we next evaluated cell death and compensatory proliferation in response to the increased inflammatory response. TUNELand Ki67-positive cells were increased in LdC-fed mice, compared with control diet-fed mice (Fig. 3L–N, Fig. S7G–I). Altogether, these results evidenced that deletion of Cdkn1a did not protect against preclinical ALD. Senescence is attenuated in Cdkn1a -/- animals fed a DuAL diet Next, we investigated the induction of senescence associated to CDKN1A. Senescence-inducing signals activate transcriptional cascades which culminate in the activation of CDKN1A/p21-p53-p16 signaling resulting in irreversible cell cycle arrest. 23 Thus, we first analyzed mechanisms of cellular senescence in Cdkn1a +/+ and Cdkn1a -/- mice, fed a DuAL diet. Interestingly, Cdkn1a -/- mice displayed a significant downregulation in CDKN1A/p21 and p53 mRNA levels compared with Cdkn1a +/+ mice (Fig. 4A,B). Although a tendency towards increased telomere shortening in Cdkn1a +/+ fed a DuAL diet, no significant changes were observed between Cdkn1a +/+ and Cdkn1a -/- mice (Fig. 4C). Senescence can be also driven by other factors including DNA damage and reactive oxygen species (ROS). Both the phosphorylation of histone H2AX (cH2AX), a marker of DNA J Cdkn1a+/+ Cdkn1a-/- ## * Chow DuAL Chow DuAL 0 2 4 6 β-GAL positive cells (%) I Chow DuAL Chow DuAL γH2AX positive cells/20 field *** n.s. ## 0 10 20 30 40 H### ** Chow DuAL Chow DuAL 0 10 20 30 4-HNE area (%) n.s. β-GAL G γH2AX F 4-HNE DuAL Cdkn1a+/+ Cdkn1a-/- ED 1.00 ± 0.10 1.37 ± 0.19 0.79 ± 0.15 1.11 ± 0.07# 1.03 ± 0.09 2.40 ±1.09 1.57 ± 0.23 2.27 ± 1.08 1.13 ± 0.34 1.82 ± 0.13 1.09 ± 0.22 1.00 ± 0.06# p16 16 PCNA 29 GAPDH 37 37 GAPDH 19 γH2AX 1.00 ± 0.07 1.19 ± 0.14 0.88 ± 0.19 0.83 ± 0.13 pRb 110 GAPDH 37 kDa Chow DuAL Cdkn1a+/+ Chow DuAL Cdkn1a-/- C Cdkn1a+/+ Cdkn1a-/- Chow DuAL Chow DuAL 0.8 1.0 1.2 1.4 1.6 Telomere/GAPDH B Chow DuAL Chow DuAL 0.0 0.5 1.0 1.5 2.0 P53 (fold change) *# A *** Chow DuAL Chow DuAL 0.0 0.5 1.0 1.5 2.0 2.5 Cdkn1a/p21 (fold change) Fig. 4. Amelioration of liver senescence is characteristic of Cdkn1a -/- mice fed a DuAL diet. Cdkn1a/p21 (A) and P53 (B) mRNA relative liver expression to Gapdh was analyzed in Cdkn1a +/+ and Cdkn1a -/- mice after DuAL diet. (C) Relative telomere length was calculated using PCR. (D) Total liver protein (two individuals’samples from each group are shown as representative of the group) were isolated from DuAL diet-fed Cdkn1a +/+ and Cdkn1a -/- mice and analyzed for cH2AX, pRb, p16, and PCNA. Protein expression levels were normalized to the levels of total GAPDH and the ratio was calculated. (E–G) Representative liver sections of Cdkn1a +/+ and Cdkn1a -/- mice stained with 4-HNE (E), cH2AX (F) and b-GAL (G). Scale bar = 100 l m. (H–J) Quantification of 4-HNE, cH2AX and b-GAL-positive cells per field. Oneway ANOVA with post hoc Tukey test was used for the calculations. n = 6–10 (intragroup: ***p<0.001, ***p<0.001; intergroup: # p<0.01, ### p<0.001). b-Gal, beta galactosidase; cH2AX, gamma H2A histone family member X; 4-HNE, 4-hydroxynonenal; Cdkn1a, cyclin-dependent kinase inhibitor 1A; DuAL, metabolic dysfunctionassociated fatty liver disease and alcohol-related liver; Gadph, glyceraldehyde-3-phosphate dehydrogenase; pRb, phospho retinoblastoma tumor suppressor; PCNA, proliferating cell nuclear antigen. JHEP Reports, --- 2025. vol. 7 j101230 7 Research article damage and 4-hydroxynonenal (4-HNE) –a marker of lipid peroxidation –revealed less cH2AX and 4-HNE expression in Cdkn1a -/- compared with Cdkn1a +/+ livers, after a DuAL diet (Fig. 4D–F,H,I,Fig. S8A,B). We further tested other markers of cellular senescence such as p16, which acts through the retinoblastoma (Rb) triggering cell cycle arrest and, therefore, decreased cell proliferation (Fig. 4D). P16, pRb, and PCNA were underexpressed in Cdkn1a -/- compared with Cdkn1a +/+ animals (Fig. 4D). Moreover, immunofluorescence for the detection of bgalactosidase (b-GAL) activity, a widely used biomarker of cellular senescence, was significantly lower in Cdkn1a -/- compared with Cdkn1a +/+ livers (Fig. 4G,J, Fig. S8C). Inflammation is reduced in Cdkn1a -/- mice in experimental MetALD Increased cellular senescence and the activation of the CDKN1A pathway act together to generate a proinflammatory environment, having a crucial role in propagating senescence and in the recruitment of immune cells to the senescent tissue. 24 In fact, Cdkn1a knockout animals showed a significant reduction in the accumulation of leukocytes (CD45 + cells) and infiltrated cells (CD11b + cells), in contrast to Cdkn1a +/+ mice, analyzed by immunofluorescence staining after a DuAL and a WD, but not after LdC diet (Fig. 5A–D, Fig. S8D–G). Furthermore, circulating F4/80 hi Ly6C hi monocytes were significantly decreased in DuAL diet-fed Cdkn1a -/- compared with Cdkn1a +/ + mice (Fig. 5E). Additionally, a clear trend towards decreased levels of mRNA transcripts for Tnf a and Il6, and significant downregulation in Il1 a and Il8 was found in Cdkn1a -/- mice, fed a DuAL diet (Fig. 5F–I). We next measured typical markers of SASP such as insoluble factors (Pge2) and non-protein molecules (Igfbp2) which showed a significant reduction in Cdkn1a -/- , compared with Cdkn1a +/+ mice (Fig. 5J,K). All these data indicated that Cdkn1a -/- mice displayed less inflammation and decreased proinflammatory secretome. Examining the function of Cdkn1a in modulating lipid metabolism in experimental models of SLD Senescence is linked to cell cycle arrest, oxidative stress, DNA damage and inflammation, and high expression of Cdkn1a, which triggers metabolic dysfunction. These alterations have been linked to promote the synthesis of genes involved in metabolism. 25 Given the protection observed in steatosis, ballooning, lobular inflammation, fibrosis, and NAS in mice with K## * Chow DuAL Chow DuAL 0 1 2 3 4 5 Igfbp4 (fold change) Cdkn1a+/+ Cdkn1a-/- J # Chow DuAL Chow DuAL 0.0 0.5 1.0 1.5 2.0 2.5 Pge2 (fold change) I## ** Chow DuAL Chow DuAL 0 2 4 6 Il8 (fold change) H### **** Chow DuAL Chow DuAL 0 1 2 3 Il1α (fold change) G Chow DuAL Chow DuAL 0.0 0.5 1.0 1.5 2.0 Il6 (fold change) 0.4020 n.s. 0.1739 Chow DuAL Chow DuAL 0 5 10 15 Tnfα (fold change) ** n.s. 0.1071 FE # * Chow DuAL Chow DuAL 0 5 10 15 20 25 Monocytes (%) * D # *** Chow DuAL Chow DuAL 0.0 0.5 1.0 1.5 CD11b positive cells (%) * C Chow DuAL Chow DuAL 0 2 4 6 CD45 positive cells (%) ** n.s. # B CD11b DuAL Cdkn1a+/+ Cdkn1a-/- A CD45 DuAL Cdkn1a+/+ Cdkn1a-/- Fig. 5. Inflammation is significantly reduced in Cdkn1a -/- mice fed a DuAL diet. (A,B) Representative immunofluorescence staining for CD45 (A) and CD11b (B) in DuAL diet-fed Cdkn1a +/+ and Cdkn1a -/- mice. Quantification of CD45- (C) and CD11b- (D) positive cells in the same samples. Arrows (/) indicate positive cells. (E) Circulating F4/80 hi Ly6C hi cells in DuAL diet-fed Cdkn1a +/+ and Cdkn1a -/- mice. Tnf a (F), Il6 (G), Il1 a (H), Il8 (I), Igfbp4 (J), and Pge2 (K) mRNA expression determined by qPCR and normalized to the amount of Gapdh in the liver of DuAL-fed mice and controls. n = 4–10; one-way ANOVA with post hoc Tukey test was used for the calculations; intragroup: **p<0.01, ****p<0.0001; intergroup: # p<0.05, ### p<0.001. Cdkn1a, cyclin-dependent kinase inhibitor 1A; DuAL, metabolic dysfunctionassociated fatty liver disease and alcohol-related liver; Igfbp4, Insulin like growth factor binding protein 4; Gadph, glyceraldehyde-3-phosphate dehydrogenase; Pge2, prostaglandin E2; qPCR, quantitative PCR; Tnf a , tumor necrosis factor alpha. 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