FAK-TRIM25 Promotes HSC Activation and Glycolysis by Inhibiting c-Myc Ubiquitination via FBXW7

FAK-TRIM25 Promotes HSC Activation and Glycolysis by Inhibiting c-Myc Ubiquitination via FBXW7 | 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 Research Article FAK-TRIM25 Promotes HSC Activation and Glycolysis by Inhibiting c-Myc Ubiquitination via FBXW7 Lu Han, Guo-Yuan Lin, Shao-Jie Chen, Qing-Xiu Zhang, Hua-Yue Wu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6993066/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 Liver fibrosis is marked by hepatic stellate cell (HSC) activation and increased glucose consumption. Focal adhesion kinase (FAK) upregulates c-Myc expression in HSCs, promoting aerobic glycolysis. This study explores how FAK promotes HSC activation and glycolysis via TRIM25. Immunohistochemistry (IHC) and Western blotting assessed the expression of FAK, TRIM25, FBXW7, c-Myc, and glycolysis-related proteins in human liver tissues and mouse models. Protein interactions were identified by co-immunoprecipitation (Co-IP) and LC-MS, and FAK and TRIM25 localization was observed by immunofluorescence. FAK inhibition reduced LX-2 cell activation, migration, and glycolysis. Co-IP and immunofluorescence confirmed FAK-TRIM25 interaction. FAK inhibits FBXW7-mediated c-Myc ubiquitination and enhances glycolysis by binding TRIM25’s RING, B-BOX, and SPRY regions. Inhibition of FAK improved liver fibrosis and glycolysis. FAK promotes HSC glycolysis through TRIM25 interaction, and its inhibition mitigates liver fibrosis, suggesting a potential therapeutic target. Focal adhesion kinase tripartite motif protein 25 aerobic glycolysis liver fibrosis FAK inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Liver fibrosis, an early stage of cirrhosis, is reversible with timely intervention[ 1 , 2 ]. Hepatic stellate cell activation leads to extracellular matrix accumulation, driving scarring. Despite the lack of approved medications for liver fibrosis, ongoing research into its pathogenesis and treatments remains vital[ 3 , 4 ]. Hepatic stellate cell (HSC) activation increases glucose utilization and upregulates enzymes for aerobic glycolysis (HK-2, PFKFB3, ENO1, PK), supporting HSC proliferation and inflammation, aiding transdifferentiation into myofibroblasts [ 5 , 6 ]. Focal adhesion kinase (FAK), a tyrosine kinase, promotes aerobic glycolysis by upregulating genes like glucose transporter protein 3 and hexokinase-2, potentially activating the PI3K/AKT pathway [ 7 – 9 ]. Our previous research showed FAK stabilizes c-Myc in LX-2 cells, but its role in liver fibrosis remains unclear [ 15 ]. We examined pY397-FAK expression in liver tissues from mice and patients with liver fibrosis using Western blotting and immunohistochemistry. Pathway changes in patient tissues were analyzed by RNA sequencing. Aerobic glycolysis in the mouse liver was assessed by measuring glycolytic enzymes, glucose, and lactate levels. Our findings suggest that FAK influences LX-2 cell activation, migration, and aerobic glycolysis. Co-IP, mass spectrometry, and immunofluorescence revealed FAK’s interaction with TRIM25 in LX-2 cells. FAK induces liver fibrosis and aerobic glycolysis via the TRIM25/FBXW7/c-Myc pathway. FAK inhibitors may offer a promising therapeutic strategy for liver fibrosis. MATERIALS AND METHODS 1. Reagents and antibodies CCl4 and corn oil were from Sigma Aldrich (USA), PF562271 from MedChemExpress (USA). Antibodies against COL1A1, smooth muscle actin, β-actin, FAK, TRIM25 from Abcam and Proteintech. PKM2, PFKFB3, HK-2, ENO1 antibodies from Chengdu Zhengneng. Plasmids for TRIM25 and FAK from GeneChem. ELISA kits from Animaluni, staining kits from Solebo, and AAV vectors from Baisheng Lejian Biotechnology. 2. Cell Culture and Transfection Methods for LX-2 and HEK 293T Cells LX-2 and HEK 293T cell lines, obtained from Wuhan Pricella, China, were authenticated by STR profiling and confirmed mycoplasma-free. Cells were cultured in high-glucose DMEM with 10% fetal bovine serum and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL) at 37°C in 5% CO₂. LX-2 cells were transfected with a lentiviral vector as a negative control, and stable cells were selected with puromycin (2-10 μg/ml). FAK overexpression was confirmed by western blot. For siRNA transfection, Lipofectamine™ 3000 and 150 pmol siRNA were mixed in Opti-MEM, incubated for 15 minutes, added to cells, and incubated for 48 hours before experimentation. The siRNA sequence is in Table S2. 3. Patient sample collection This study included 30 liver fibrosis or cirrhosis patients and 8 controls, with clinical details in Table S1. Exclusion criteria were diabetes, liver glycogen deposition, or recent insulin/hypoglycemic drug use. Liver samples were obtained from patients undergoing partial hepatectomy. Informed consent was obtained from all participants, and the study was approved by the Clinical Trial Ethics Committee of Guizhou Medical University (2023 lunshen 401). 4. Experimental protocol Forty-two male C57BL/6J mice were divided into seven groups: control, CCL4 model, inhibitor model (CCL4+PF562271), virus control (AAV-vector), TRIM25 overexpression (AAV-TRIM25), virus model (AAV-TRIM25+CCL4), and composite model (AAV-TRIM25+CCL4+PF562271). Four animals from the virus control and TRIM25 groups were used for expression validation. Mice received 10% CCL4 (15μl/g) thrice weekly for six weeks. The inhibitor and composite models received PF562271 (14 mg/kg) orally for four weeks, based on previous studies[16]. Body weights were recorded weekly. 5. RT-qPCR Total RNA was isolated using a RNA kit, and subsequently cDNA was synthesized with a cDNA synthesis kit. The cDNA was amplified using specific primers, with β-actin acting as the reference. The relative expression was quantified as 2-ΔΔCq compared to β-actin.Primer sequences for each gene are shown in TableS3. 6. Western blot Proteins were extracted from LX-2 cells using a total protein extraction kit, and concentration was determined with a BCA kit. Samples were incubated overnight with primary antibody at 4°C, followed by secondary antibody for 2 hours at room temperature. Protein expression was analyzed using an ECL kit. 7. Histopathological studies and Immunohistochemistry (IHC) staining Liver tissue from patients and mice was fixed in 4% formaldehyde, embedded in paraffin, and sectioned (4–5 μm). Sections were stained with hematoxylin-eosin, Sirius red, and Masson’s trichrome. Immunohistochemistry (IHC) was performed as previously described, and images were captured using a Zeiss Axio imaging system. 8. Inflammation assessment 9. Mouse plasma supernatant was collected, and inflammatory markers (LPS, TNF-α, IL-1, IL-6) were detected using an ELISA kit following the manufacturer’s instructions. 10. Liver function The levels of AST and ALT in plasma samples were measured using automated techniques with a CD-1600CS biochemistry analyzer from Abbott Laboratories (Abbott Park, IL, USA). 11. Wound healing assay and Transwell assays LX-2 cells were plated in 6-well dishes to assess migration via wound healing and transwell assays. For the wound healing assay, cells were allowed to reach confluency, a line was drawn, and migration was observed at 0 and 72 hours with serum-free DMEM. For transwell assays, cells were seeded in the upper chamber with serum-free DMEM, and migration was assessed after 6 hours by staining and counting cells in five random fields. 12. Plasmid transfection HEK 293T cells at 70% confluency were transfected using PEI-40K. After 20 minutes in Opti-MEM, the DNA-PEI complex was added and incubated for 6 hours in a 5% CO₂ environment. The medium was replaced, and cells were incubated for 48 hours before protein extraction for further analysis. 13. Immunoprecipitation Proteins were extracted from LX-2 and HEK 293T cells at 80% confluency. Cells were lysed, and proteins were categorized into IP, IgG, and Input groups. The IP group received specific antibodies, while IgG served as a negative control. After overnight centrifugation at 4°C, Protein A/G beads were added, washed, boiled with loading buffer, and stored at -80°C for Western Blot analysis. 14. Immunofluorescence Healthy LX-2 cells were cultured on chamber slides for immunofluorescence. After fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton-X-100, cells were sealed with goat serum and incubated with primary antibody overnight at 4°C. After PBS washing, the secondary antibody was applied, followed by DAPI staining. Samples were imaged under a fluorescence microscope. 15. Glucose and lactate assay The supernatant from each group of LX-2 cells was collected and stored at -80°C. Mouse liver tissue (40 mg) from each group was homogenised with PBS, and lactate levels and protein concentrations were measured. Lactate and glucose were detected according to the instructions. 16. RNA sequencing, differential gene analysis, and enrichment analysis Liver tissues from 30 liver fibrosis patients and 8 controls were analyzed using Illumina transcriptome sequencing. Genes with an FDR ≤ 0.05 and |log2FC| ≥ 1 were identified as differentially expressed. GO annotation and KEGG enrichment analyses were conducted to determine their biological significance. 17. Statistical analysis Statistical analyses were performed using GraphPad Prism (version 9.0). Data are presented as mean ± SD. Normality and homogeneity of variances were tested using Shapiro-Wilk and Levene's tests. Student's t-test or one-way ANOVA with post hoc tests (Tukey's or Games-Howell) were used for comparisons. Non-parametric tests (Mann-Whitney U or Kruskal-Wallis) were applied for data not meeting normality assumptions. A significance level of **p < 0.05** was used. RESULTS 1. Liver tissue from patients with liver fibrosis shows a higher level of FAK expression. Liver tissue samples from 30 liver fibrosis patients and 8 controls were analyzed. Fibrosis tissues showed disrupted lobular structure, collagen deposition (Masson/Sirius red staining), and elevated pY397-FAK expression (Figure 1A-B). Western blot revealed increased COL1A1 and α-SMA levels (Figure 1C). RNA sequencing and KEGG/GO analyses identified NF-κB signaling and inflammation pathways, with FAK as a key molecule (Figure 1D-E). These findings link FAK to liver fibrosis progression. 2. By inhibiting FAK, liver fibrosis and aerobic glycolysis can be alleviated in mice. In a liver fibrosis mouse model, FAK inhibitor treatment reduced fibrosis, ALT, AST levels (P < 0.01), and COL1A1/α-SMA expression (Figure 2A-B). pY397-FAK levels decreased, along with glucose and lactate production (Figure 2C-D). Inflammatory factors (IL-1β, IL-6, TNF-α, LPS) were also lower (P < 0.05) (Figure S1), suggesting FAK inhibition alleviates liver fibrosis, inflammation, and glycolysis. 3. FAK affects hepatic stellate cell activation, migration, and aerobic glycolysis FAK's effects on LX-2 cell activation and glycolysis were studied using FAK inhibitor (PF562271) and overexpression. FAK inhibition reduced COL1A1 and α-SMA expression, while overexpression increased these markers (Figure 3A). Western blot analysis showed that FAK inhibition decreased glycolytic enzyme levels, while overexpression increased them (Figure 3C). FAK inhibition reduced cell migration and glucose uptake, while overexpression enhanced migration, glucose uptake, and lactate production (Figure 3B, 3D-E). These results suggest FAK regulates LX-2 cell activation, migration, and aerobic glycolysis. 4. FAK has the potential to alter the stability of c-Myc and TRIM25 proteins. WB and RT-qPCR experiments revealed that c-Myc protein expression decreased with increasing FAK inhibitor concentrations, while FAK overexpression increased c-Myc levels (Figure 4A). Despite reduced FAK levels, c-Myc mRNA expression remained unchanged, indicating post-transcriptional regulation (Figure 4B). CHX experiments showed that FAK inhibitor reduced c-Myc protein stability (Figure 4C). Mass spectrometry identified TRIM25, which stabilizes c-Myc via ubiquitination (Figure 4E). Further analysis confirmed that TRIM25 protein stability was reduced in the FAK inhibitor group, suggesting FAK stabilizes c-Myc protein through TRIM25 (Figure 4D-E). 5. FAK interacts with TRIM25 and increases FBXW7 ubiquitination FAK and TRIM25 interaction was confirmed through immunoprecipitation, showing a direct binding (Figure 5A) and colocalization in the nucleus and cytoplasm (Figure 5B). Co-transfection in HEK 293T cells validated their interaction (Figure 5C). FAK inhibitor treatment did not affect TRIM25 mRNA levels (Figure 5D). Truncated TRIM25 vectors identified the SPRY region as crucial for binding (Figure 5E). FAK enhances FBXW7 ubiquitination and c-Myc stabilization (Figure 5F). FAK overexpression increased TRIM25 levels and decreased FBXW7 (Figure 6A), with IHC and WB results confirming these findings in mouse liver (Figure 6B-C). TRIM25 and c-Myc were elevated in liver fibrosis patients (Figure 6D). These results suggest FAK regulates the TRIM25/FBXW7/c-Myc pathway in liver fibrosis. 6. TRIM25 is involved in promoting hepatic stellate cell activation and aerobic glycolysis by interacting with FAK. TRIM25 was silenced in FAK-overexpressing LX-2 cells to explore its role in FAK's functions. WB analysis showed that TRIM25 knockdown restored the TRIM25/p-Tyr/FBXW7/c-Myc pathway (Figure 7A) and reduced COL1A1 expression (Figure 7C), along with decreased levels of aerobic glycolysis enzymes (HK2, PKM2, ENO1, and PFKFB3) (Figure 7B). Additionally, TRIM25 knockdown impaired LX-2 cell migration and activation (Figure 7D-E) and reduced glucose uptake and lactate production (Figure 7F-J). These results suggest that FAK regulates aerobic glycolysis in LX-2 cells through TRIM25. 7. FAK exacerbates liver fibrosis and aerobic glycolysis in mice by phosphorylating TRIM25 AAV-Trim25 was administered to mice for 4 weeks to investigate FAK's role in c-Myc regulation via the TRIM25/FBXW7 pathway in liver fibrosis. Liver analysis showed TRIM25 overexpression without inflammation, but it exacerbated fibrosis. FAK inhibition alleviated fibrosis, reduced inflammatory markers (Figure S5), and reversed c-Myc expression (Figure 8E). Aerobic glycolysis markers were elevated in the viral model (Figure 8C, 8D), while FAK inhibition reversed these effects. These findings suggest TRIM25 exacerbates liver fibrosis and glycolysis, and FAK inhibition can mitigate these effects (Figure 8A, 8B, 8F). DISCUSSION This study investigates how FAK regulates stellate cell activation, migration, and glycolysis via TRIM25 in liver fibrosis. Our results show FAK upregulation promotes LX-2 activation and glycolysis. Co-IP revealed FAK stabilizes TRIM25 through tyrosine phosphorylation, enhancing FBXW7 ubiquitination and c-Myc stability, increasing ENO1 transcription. In vivo tests suggest FAK inhibitors may help manage fibrosis progression. This highlights a novel FAK-TRIM25 interaction in liver fibrosis [ 15 , 19 ]. FAK, a cytoplasmic tyrosine kinase, is highly expressed in invasive cancers and promotes aerobic glycolysis in tumors [ 11 , 12 , 13 ]. Our study supports this, showing elevated FAK and aerobic glycolytic enzyme expression in liver tissues of fibrosis patients. FAK is enriched in the adhesion pathway, correlating with liver dysfunction and bile acid metabolism disruption, suggesting its potential as a clinical target. FAK interacts with TRIM25, an E3 ubiquitin ligase that stabilizes c-Myc and promotes cancer metastasis [ 19 ]. FAK regulates the TRIM25/FBXW7/c-Myc signalling pathway, promoting liver fibrosis and aerobic glycolysis in LX-2 cells and mice. It stabilises TRIM25 through post-translational modification, reduces its self-ubiquitination, and enhances the stability of c-Myc [ 18 ]. In vivo, FAK inhibitors block this pathway, alleviating liver fibrosis and aerobic glycolysis. These findings suggest that FAK inhibitors may be a potential therapeutic approach for liver fibrosis, and that TRIM25 is a key mediator in FAK-driven fibrotic progression. In summary, our study shows that FAK promotes liver fibrosis and aerobic glycolysis at cellular, animal, and tissue levels. Specific FAK inhibitors could mitigate these processes. FAK regulates the TRIM25/FBXW7/c-Myc pathway by binding to TRIM25’s RING, BOX, and SPRY regions, enhancing liver fibrosis and glycolysis. Despite limitations, such as using one hepatic stellate cell type, our findings lay the foundation for further exploration of liver fibrosis causes and management. Declarations Ethics approval and consent to participate The study was approved by the Institutional Review Board of The Affiliated Hospital of Guizhou Medical University (2023 Lunshen 401). Written informed consent was obtained from all participants. All experiments, including those involving mice, complied with ethical regulations and were approved by the Animal Ethics Committee of Guizhou Medical University (No. 2403086). Consent for publication Not applicable. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare no conflicts. Funding This work was supported by the Science and Technology Program of the Guizhou Province (No. [2021] 094.), the National Natural Science Foundation of China (No.82060116 and No.82260129), the Guizhou Provincial Science and Technology Program (QKH JC-ZK [2023]-214) and The Doctoral Research Start-up Fund Project of Guizhou Medical University Affiliated Hospital (No.gyfybsky[2021]-63 and No.gyfybsky-2025-10) Authors' contributions All authors contributed to the conception and design of the study. Xue-Ke Zhao designed and supervised the study, and reviewed the final manuscript. Lu Han performed the majority of the experiments and drafted the initial manuscript. Fan Lu contributed to revising the manuscript. Gao-Liang Zou was responsible for conducting the WB experiments. Qing-Xiu Zhang, Ya Zhang, and Tao Ran conducted the animal experiments. Tao Huang performed statistical analysis. Hong-Fei Pu and Jing-Lin Wang collected the data. Jian-Chao Li assisted with experimental design. Hua-Yue Wu assisted with experimental procedures. All authors read and approved the final manuscript. Acknowledgements We thank Bullet Edits Limited for the linguistic editing and proofreading of the manuscript.We would like to thank Guixiu Xie and Shuqiang Han for their support of the project. References Zubiete-Franco I, Fernández‐Tussy P, Barbier‐Torres L, Simon J, Fernández‐Ramos D, Lopitz‐Otsoa F, et al. Deregulated neddylation in liver fibrosis. Hepatology. 2017;65(2):694–709. Oh Y, Park O, Swierczewska M, Hamilton JP, Park JS, Kim TH, et al. Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells. Hepatology. 2016;64(1):209–23. Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol. 2017;14(7):397–411. Caviglia JM, Yan J, Jang MK, Gwak GY, Affo S, Yu L, et al. MicroRNA-21 and Dicer are dispensable for hepatic stellate cell activation and the development of liver fibrosis. Hepatology. 2018;67(6):2414–29. Trivedi P, Wang S, Friedman SL. The Power of Plasticity—Metabolic Regulation of Hepatic Stellate Cells. Cell Metabol. 2021;33(2):242–57. Mejias M, Gallego J, Naranjo-Suarez S, Ramirez M, Pell N, Manzano A, et al. CPEB4 Increases Expression of PFKFB3 to Induce Glycolysis and Activate Mouse and Human Hepatic Stellate Cells, Promoting Liver Fibrosis. Gastroenterology. 2020;159(1):273–88. Golubovskaya V. Targeting FAK in human cancer: from finding to first clinical trials. Front Biosci. 2014;19(4):687. Raab M, Lu Y, Kohler K, Smith X, Strebhardt K, Rudd CE. LFA-1 activates focal adhesion kinases FAK1/PYK2 to generate LAT-GRB2-SKAP1 complexes that terminate T-cell conjugate formation. Nat Commun. 2017;8(1):16001. 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The MAP3K13-TRIM25-FBXW7alpha axis affects c-Myc protein stability and tumor development. Cell Death Differ. 2020;27(2):420–33. Qie S, Majumder M, Mackiewicz K, Howley BV, Peterson YK, Howe PH et al. Fbxo4-mediated degradation of Fxr1 suppresses tumorigenesis in head and neck squamous cell carcinoma. Nat Commun. 2017;8(1). Supplementary Files TableS1.docx TableS2.docx TableS3.docx supplementFigure.docx 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. We do this by developing innovative software and high quality services for the global research community. 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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-6993066","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481228432,"identity":"2e355c0e-6070-4352-8a03-e9ceaf8846bb","order_by":0,"name":"Lu Han","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Han","suffix":""},{"id":481228433,"identity":"5e51a332-566a-409f-b616-05c66ac961ba","order_by":1,"name":"Guo-Yuan Lin","email":"","orcid":"","institution":"The Affiliated Hospital of Guizhou Medical 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15:44:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6993066/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6993066/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86537109,"identity":"f905a859-b731-4c36-abce-2c563833bf6f","added_by":"auto","created_at":"2025-07-11 18:56:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2263658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated FAK expression in liver fibrosis patients, enriched in the adhesion kinase pathway.\u003c/strong\u003e (A) HE, Masson, and Sirius red staining show differences in liver tissue between the two groups. (B) IHC detected hepatic FAK expression in liver fibrosis patients (n=10) and controls (n=8). (C) WB analysis of α-SMA, COL1A1, and p-FAK expression in liver fibrosis patients (n=10) and controls (n=8). Liver tissues from 30 liver fibrosis patients and 8 controls were analyzed by RNA sequencing and WB. (D-E) KEGG and GO enrichment analysis bubble diagram. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001. Data represent mean ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/6345947b6e7a5fc4845c34b8.png"},{"id":86537415,"identity":"12471ead-767b-4bfd-8f51-80f7ca5d241e","added_by":"auto","created_at":"2025-07-11 19:04:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":769542,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAK is elevated in liver tissues of liver fibrosis mice, and inhibition of FAK can mitigate fibrosis and aerobic glycolysis in liver fibrosis mice. \u003c/strong\u003e(A) Differences in liver tissues of different groups of mice were observed by HE, Masson, and Sirius red staining (n = 5 mice/group) and expression of ALT and AST in different groups of mice. (B-C,E) Expression of liver fibrosis markers, α-SMA and COL1A1, and the expression of pY397-FAK and FAK were detected. (D) Glucose and lactate content of liver tissues from three groups of mice were measured. (F)The aerobic glycolytic enzyme expression in different groups of mice was detected by WB. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.Data shown are mean ± SD of three independent experiments\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/c6abba88b9d41af7ac5f3c79.png"},{"id":86537901,"identity":"1f01e455-27d2-4f79-b1ec-7062b1d5dd04","added_by":"auto","created_at":"2025-07-11 19:12:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":972923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAK affects activation, migration, and aerobic glycolysis of hepatic stellate cells.\u003c/strong\u003e (A) Following the overexpression or inhibition of p-FAK, the expression levels of the liver fibrosis markers COL1A1 and α-SMA were detected using Western blot. (B) Glucose and lactate levels in LX-2 cells were measured by collecting supernatants. (C) Aerobic glycolytic enzymes were detected by WB. (D) Migration ability was assessed by transwell assay (n = 3). (E) Migration ability was further evaluated by cell scratch assay (n = 3). *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001. Data represent mean ± SD of three independent experiments.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/85507ff0d43a3d16ee03d3a6.png"},{"id":86537113,"identity":"a6f15145-9301-4550-9b73-e0ee797a497b","added_by":"auto","created_at":"2025-07-11 18:56:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1013023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAK affects c-Myc and TRIM25 protein stability.\u003c/strong\u003eAfter constructing the FAK inhibition/overexpression model using PF562271 (FAK inhibitor) and lentiviral overexpression, (A-B) c-Myc protein and mRNA expression were analyzed by WB and RT-qPCR, with data as mean ± SD, ns P \u0026gt; 0.05. (C) Protein synthesis was blocked by CHX, and Myc expression was quantified by densitometry. (D) FAK affects TRIM25 protein stability, measured by WB after CHX treatment. (E) FAK interacts with TRIM25; after FAK overexpression in LX-2 cells, FLAG-purified proteins were analyzed by SDS-PAGE and mass spectrometry. Data are mean ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/c8c8d12811b83399cb30f0f3.png"},{"id":86537418,"identity":"288460e8-6ad4-449e-8565-dab42717a8f1","added_by":"auto","created_at":"2025-07-11 19:04:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2223536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAK interacts with TRIM25 and enhances FBXW7 ubiquitination, with the RING, BOX, and SPRY regions of TRIM25 critical for binding to FAK.\u003c/strong\u003e(A) Co-IP shows FAK interacting with TRIM25 in LX-2 cells. (B) FAK and TRIM25 co-localize in the cytoplasm and nucleus. (C) Exogenous Co-IP confirms the interaction. (D) RT-qPCR analysis of TRIM25 mRNA expression. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001. (E) Schematic of TRIM25 structure and truncated vectors, highlighting key regions for FAK binding. (F) Co-transfection in 293T cells shows FAK’s effect on FBXW7 ubiquitination. Data are mean ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/64b6c11d50cc13abaa7ae916.png"},{"id":86537111,"identity":"f8c8fdfc-af2b-46ba-9bfa-7752de151114","added_by":"auto","created_at":"2025-07-11 18:56:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2101917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAK can regulate the TRIM25/FBXW7/c-Myc pathway by phosphorylating TRIM25.\u003c/strong\u003e(A) Protein changes in the TRIM25/p-Tyr/FBXW7 pathway were detected by WB in different groups. (B) TRIM25 and c-Myc protein expression were detected by IHC in liver tissues of mice in all groups. (C) Alterations in the FAK/TRIM25/p-Tyr/FBXW7/c-Myc pathway. (D-E) Detection of p-FAK, TRIM25, FBXW7, and c-Myc protein alterations by IHC and WB in liver fibrosis and control groups.Data shown are mean ± SD of three independent experiments\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/8f63cc55928a121e659936f9.png"},{"id":86537417,"identity":"a5ea6d2d-9ba4-46ce-ac3d-d350880bfffb","added_by":"auto","created_at":"2025-07-11 19:04:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2180737,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM25 is involved in FAK-mediated effects on LX-2 cell activation, migration, and aerobic glycolysis. \u003c/strong\u003e(A) In LX-2 cells overexpressing FAK, TRIM25 was knocked down, and protein expression of p-FAK, TRIM25, p-Tyr, FBXW7, and c-Myc was detected by WB. (B) Aerobic glycolysis enzyme expression was measured by WB. (C) COL1A1 expression was detected after TRIM25 knockdown. (D) LX-2 cell migration was assessed by transwell assay. (E) Migration was also tested by the cell scratch method. (F-J) Lactate and glucose levels in cell supernatants were measured. Data are mean ± SD from three independent experiments, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/35992359b528faba62ce178e.png"},{"id":86537115,"identity":"86033e29-5e6e-45a2-99ee-ce4c04cc2b63","added_by":"auto","created_at":"2025-07-11 18:56:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1460148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAK exacerbates liver fibrosis and aerobic glycolysis in mice by modulating TRIM25. TRIM25 was overexpressed via tail vein injection, followed by CCL4-induced liver fibrosis modeling (n=5/group). \u003c/strong\u003e(A) α-SMA expression was detected by WB. (B) Liver tissues were stained with HE, Masson, and Sirius red. (C) Aerobic glycolysis enzyme expression was measured by WB. (D) Glucose and lactate levels were detected. (E-F) Protein expression of FAK, p-FAK, TRIM25, FBXW7, and c-Myc was analyzed. Data are mean ± SD from three independent experiments.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/7a354a583227c391d047cc5c.png"},{"id":87872202,"identity":"0fe61577-dd0a-44ae-b779-2fa4b7a842b2","added_by":"auto","created_at":"2025-07-30 00:42:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13700471,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/f1d28eff-7060-4e45-ace5-a802f1e8a5fc.pdf"},{"id":86537116,"identity":"6c1d05b9-519c-427b-98ab-92477f1ee6be","added_by":"auto","created_at":"2025-07-11 18:56:04","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":14587,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/7f4391b8b4d80d0c5e033b18.docx"},{"id":86537121,"identity":"97acb2af-2c64-49c8-abff-49957f815592","added_by":"auto","created_at":"2025-07-11 18:56:04","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":12176,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/a731a51d479a97b6f7f9509b.docx"},{"id":86537902,"identity":"0c8694f2-3d23-4880-b1f3-a06ecf4b4044","added_by":"auto","created_at":"2025-07-11 19:12:04","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":11951,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/eb656aa310c3a442c535ef5a.docx"},{"id":86537425,"identity":"95b78387-abaa-4039-afea-8e81369a2992","added_by":"auto","created_at":"2025-07-11 19:04:04","extension":"docx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":8312645,"visible":true,"origin":"","legend":"","description":"","filename":"supplementFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6993066/v1/086f50d230be53a6b9b2b945.docx"}],"financialInterests":"","formattedTitle":"FAK-TRIM25 Promotes HSC Activation and Glycolysis by Inhibiting c-Myc Ubiquitination via FBXW7","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLiver fibrosis, an early stage of cirrhosis, is reversible with timely intervention[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Hepatic stellate cell activation leads to extracellular matrix accumulation, driving scarring. Despite the lack of approved medications for liver fibrosis, ongoing research into its pathogenesis and treatments remains vital[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHepatic stellate cell (HSC) activation increases glucose utilization and upregulates enzymes for aerobic glycolysis (HK-2, PFKFB3, ENO1, PK), supporting HSC proliferation and inflammation, aiding transdifferentiation into myofibroblasts [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Focal adhesion kinase (FAK), a tyrosine kinase, promotes aerobic glycolysis by upregulating genes like glucose transporter protein 3 and hexokinase-2, potentially activating the PI3K/AKT pathway [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our previous research showed FAK stabilizes c-Myc in LX-2 cells, but its role in liver fibrosis remains unclear [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe examined pY397-FAK expression in liver tissues from mice and patients with liver fibrosis using Western blotting and immunohistochemistry. Pathway changes in patient tissues were analyzed by RNA sequencing. Aerobic glycolysis in the mouse liver was assessed by measuring glycolytic enzymes, glucose, and lactate levels. Our findings suggest that FAK influences LX-2 cell activation, migration, and aerobic glycolysis. Co-IP, mass spectrometry, and immunofluorescence revealed FAK\u0026rsquo;s interaction with TRIM25 in LX-2 cells. FAK induces liver fibrosis and aerobic glycolysis via the TRIM25/FBXW7/c-Myc pathway. FAK inhibitors may offer a promising therapeutic strategy for liver fibrosis.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e1.\u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eReagents and antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCCl4 and corn oil were from Sigma Aldrich (USA), PF562271 from MedChemExpress (USA). Antibodies against COL1A1, smooth muscle actin, β-actin, FAK, TRIM25 from Abcam and Proteintech. PKM2, PFKFB3, HK-2, ENO1 antibodies from Chengdu Zhengneng. Plasmids for TRIM25 and FAK from GeneChem. ELISA kits from Animaluni, staining kits from Solebo, and AAV vectors from Baisheng Lejian Biotechnology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u0026nbsp; \u0026nbsp;Cell Culture and Transfection Methods for LX-2 and HEK 293T Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLX-2 and HEK 293T cell lines, obtained from Wuhan Pricella, China, were authenticated by STR profiling and confirmed mycoplasma-free. Cells were cultured in high-glucose DMEM with 10% fetal bovine serum and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL) at 37°C in 5% CO₂. LX-2 cells were transfected with a lentiviral vector as a negative control, and stable cells were selected with puromycin (2-10 μg/ml). FAK overexpression was confirmed by western blot. For siRNA transfection, Lipofectamine™ 3000 and 150 pmol siRNA were mixed in Opti-MEM, incubated for 15 minutes, added to cells, and incubated for 48 hours before experimentation. The siRNA sequence is in Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u0026nbsp; \u0026nbsp;Patient sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included 30 liver fibrosis or cirrhosis patients and 8 controls, with clinical details in Table S1. Exclusion criteria were diabetes, liver glycogen deposition, or recent insulin/hypoglycemic drug use. Liver samples were obtained from patients undergoing partial hepatectomy. Informed consent was obtained from all participants, and the study was approved by the Clinical Trial Ethics Committee of Guizhou Medical University (2023 lunshen 401).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u0026nbsp; \u0026nbsp;Experimental protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty-two male C57BL/6J mice were divided into seven groups: control, CCL4 model, inhibitor model (CCL4+PF562271), virus control (AAV-vector), TRIM25 overexpression (AAV-TRIM25), virus model (AAV-TRIM25+CCL4), and composite model (AAV-TRIM25+CCL4+PF562271). Four animals from the virus control and TRIM25 groups were used for expression validation. Mice received 10% CCL4 (15μl/g) thrice weekly for six weeks. The inhibitor and composite models received PF562271 (14 mg/kg) orally for four weeks, based on previous studies[16]. Body weights were recorded weekly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.\u0026nbsp; \u0026nbsp;RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using a RNA kit, and subsequently cDNA was synthesized with a cDNA synthesis kit. The cDNA was amplified using specific primers, with β-actin acting as the reference. The relative expression was quantified as 2-ΔΔCq compared to β-actin.Primer sequences for each gene are shown in TableS3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.\u0026nbsp; \u0026nbsp;Western blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from LX-2 cells using a total protein extraction kit, and concentration was determined with a BCA kit. Samples were incubated overnight with primary antibody at 4°C, followed by secondary antibody for 2 hours at room temperature. Protein expression was analyzed using an ECL kit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7.\u0026nbsp; \u0026nbsp; Histopathological studies and Immunohistochemistry (IHC) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver tissue from patients and mice was fixed in 4% formaldehyde, embedded in paraffin, and sectioned (4–5\u0026nbsp;μm). Sections were stained with hematoxylin-eosin, Sirius red, and Masson’s trichrome. Immunohistochemistry (IHC) was performed as previously described, and images were captured using a Zeiss Axio imaging system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8.\u0026nbsp; \u0026nbsp;Inflammation assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e9.\u0026nbsp; \u0026nbsp;Mouse plasma supernatant was collected, and inflammatory markers (LPS, TNF-α, IL-1, IL-6) were detected using an ELISA kit following the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10.\u0026nbsp; Liver function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe levels of AST and ALT in plasma samples were measured using automated techniques with a CD-1600CS biochemistry analyzer from Abbott Laboratories (Abbott Park, IL, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e11.\u0026nbsp;Wound healing assay and Transwell assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLX-2 cells were plated in 6-well dishes to assess migration via wound healing and transwell assays. For the wound healing assay, cells were allowed to reach confluency, a line was drawn, and migration was observed at 0 and 72 hours with serum-free DMEM. For transwell assays, cells were seeded in the upper chamber with serum-free DMEM, and migration was assessed after 6 hours by staining and counting cells in five random fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e12.\u0026nbsp; Plasmid transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK 293T cells at 70% confluency were transfected using PEI-40K. After 20 minutes in Opti-MEM, the DNA-PEI complex was added and incubated for 6 hours in a 5% CO₂ environment. The medium was replaced, and cells were incubated for 48 hours before protein extraction for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e13.\u0026nbsp; Immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from LX-2 and HEK 293T cells at 80% confluency. Cells were lysed, and proteins were categorized into IP, IgG, and Input groups. The IP group received specific antibodies, while IgG served as a negative control. After overnight centrifugation at 4°C, Protein A/G beads were added, washed, boiled with loading buffer, and stored at -80°C for Western Blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e14.\u0026nbsp; Immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHealthy LX-2 cells were cultured on chamber slides for immunofluorescence. After fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton-X-100, cells were sealed with goat serum and incubated with primary antibody overnight at 4°C. After PBS washing, the secondary antibody was applied, followed by DAPI staining. Samples were imaged under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e15.\u0026nbsp; Glucose and lactate assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supernatant from each group of LX-2 cells was collected and stored at -80°C. Mouse liver tissue (40 mg) from each group was homogenised with PBS, and lactate levels and protein concentrations were measured. Lactate and glucose were detected according to the instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e16.\u0026nbsp;RNA sequencing, differential gene analysis, and enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver tissues from 30 liver fibrosis patients and 8 controls were analyzed using Illumina transcriptome sequencing. Genes with an FDR ≤ 0.05 and |log2FC| ≥ 1 were identified as differentially expressed. GO annotation and KEGG enrichment analyses were conducted to determine their biological significance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e17.\u0026nbsp;Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism (version 9.0). Data are presented as mean ± SD. Normality and homogeneity of variances were tested using Shapiro-Wilk and Levene's tests. Student's t-test or one-way ANOVA with post hoc tests (Tukey's or Games-Howell) were used for comparisons. Non-parametric tests (Mann-Whitney U or Kruskal-Wallis) were applied for data not meeting normality assumptions. A significance level of **p \u0026lt; 0.05** was used.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e1. Liver tissue from patients with liver fibrosis shows a higher level of FAK expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver tissue samples from 30 liver fibrosis patients and 8 controls were analyzed. Fibrosis tissues showed disrupted lobular structure, collagen deposition (Masson/Sirius red staining), and elevated pY397-FAK expression (Figure 1A-B). Western blot revealed increased COL1A1 and α-SMA levels (Figure 1C). RNA sequencing and KEGG/GO analyses identified NF-κB signaling and inflammation pathways, with FAK as a key molecule (Figure 1D-E). These findings link FAK to liver fibrosis progression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. By inhibiting FAK, liver fibrosis and aerobic glycolysis can be alleviated in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a liver fibrosis mouse model, FAK inhibitor treatment reduced fibrosis, ALT, AST levels (P \u0026lt; 0.01), and COL1A1/α-SMA expression (Figure 2A-B). pY397-FAK levels decreased, along with glucose and lactate production (Figure 2C-D). Inflammatory factors (IL-1β, IL-6, TNF-α, LPS) were also lower (P \u0026lt; 0.05) (Figure S1), suggesting FAK inhibition alleviates liver fibrosis, inflammation, and glycolysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. FAK affects hepatic stellate cell activation, migration, and aerobic glycolysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFAK's effects on LX-2 cell activation and glycolysis were studied using FAK inhibitor (PF562271) and overexpression. FAK inhibition reduced COL1A1 and\u0026nbsp;α-SMA expression, while overexpression increased these markers (Figure 3A). Western blot analysis showed that FAK inhibition decreased glycolytic enzyme levels, while overexpression increased them (Figure 3C). FAK inhibition reduced cell migration and glucose uptake, while overexpression enhanced migration, glucose uptake, and lactate production (Figure 3B, 3D-E). These results suggest FAK regulates LX-2 cell activation, migration, and aerobic glycolysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. FAK has the potential to alter the stability of c-Myc and TRIM25 proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWB and RT-qPCR experiments revealed that c-Myc protein expression decreased with increasing FAK inhibitor concentrations, while FAK overexpression increased c-Myc levels (Figure 4A). Despite reduced FAK levels, c-Myc mRNA expression remained unchanged, indicating post-transcriptional regulation (Figure 4B). CHX experiments showed that FAK inhibitor reduced c-Myc protein stability (Figure 4C). Mass spectrometry identified TRIM25, which stabilizes c-Myc via ubiquitination (Figure 4E). Further analysis confirmed that TRIM25 protein stability was reduced in the FAK inhibitor group, suggesting FAK stabilizes c-Myc protein through TRIM25 (Figure 4D-E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. \u0026nbsp;FAK interacts with TRIM25 and increases FBXW7 ubiquitination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFAK and TRIM25 interaction was confirmed through immunoprecipitation, showing a direct binding (Figure 5A) and colocalization in the nucleus and cytoplasm (Figure 5B). Co-transfection in HEK 293T cells validated their interaction (Figure 5C). FAK inhibitor treatment did not affect TRIM25 mRNA levels (Figure 5D). Truncated TRIM25 vectors identified the SPRY region as crucial for binding (Figure 5E). FAK enhances FBXW7 ubiquitination and c-Myc stabilization (Figure 5F). FAK overexpression increased TRIM25 levels and decreased FBXW7 (Figure 6A), with IHC and WB results confirming these findings in mouse liver (Figure 6B-C). TRIM25 and c-Myc were elevated in liver fibrosis patients (Figure 6D). These results suggest FAK regulates the TRIM25/FBXW7/c-Myc pathway in liver fibrosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. TRIM25 is involved in promoting hepatic stellate cell activation and aerobic glycolysis by interacting with FAK.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTRIM25 was silenced in FAK-overexpressing LX-2 cells to explore its role in FAK's functions. WB analysis showed that TRIM25 knockdown restored the TRIM25/p-Tyr/FBXW7/c-Myc pathway (Figure 7A) and reduced COL1A1 expression (Figure 7C), along with decreased levels of aerobic glycolysis enzymes (HK2, PKM2, ENO1, and PFKFB3) (Figure 7B). Additionally, TRIM25 knockdown impaired LX-2 cell migration and activation (Figure 7D-E) and reduced glucose uptake and lactate production (Figure 7F-J). These results suggest that FAK regulates aerobic glycolysis in LX-2 cells through TRIM25.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. FAK exacerbates liver fibrosis and aerobic glycolysis in mice by phosphorylating TRIM25\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAAV-Trim25 was administered to mice for 4 weeks to investigate FAK's role in c-Myc regulation via the TRIM25/FBXW7 pathway in liver fibrosis. Liver analysis showed TRIM25 overexpression without inflammation, but it exacerbated fibrosis. FAK inhibition alleviated fibrosis, reduced inflammatory markers (Figure S5), and reversed c-Myc expression (Figure 8E). Aerobic glycolysis markers were elevated in the viral model (Figure 8C, 8D), while FAK inhibition reversed these effects. These findings suggest TRIM25 exacerbates liver fibrosis and glycolysis, and FAK inhibition can mitigate these effects (Figure 8A, 8B, 8F).\u0026nbsp;\u003c/p\u003e\n"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study investigates how FAK regulates stellate cell activation, migration, and glycolysis via TRIM25 in liver fibrosis. Our results show FAK upregulation promotes LX-2 activation and glycolysis. Co-IP revealed FAK stabilizes TRIM25 through tyrosine phosphorylation, enhancing FBXW7 ubiquitination and c-Myc stability, increasing ENO1 transcription. In vivo tests suggest FAK inhibitors may help manage fibrosis progression. This highlights a novel FAK-TRIM25 interaction in liver fibrosis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFAK, a cytoplasmic tyrosine kinase, is highly expressed in invasive cancers and promotes aerobic glycolysis in tumors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our study supports this, showing elevated FAK and aerobic glycolytic enzyme expression in liver tissues of fibrosis patients. FAK is enriched in the adhesion pathway, correlating with liver dysfunction and bile acid metabolism disruption, suggesting its potential as a clinical target. FAK interacts with TRIM25, an E3 ubiquitin ligase that stabilizes c-Myc and promotes cancer metastasis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFAK regulates the TRIM25/FBXW7/c-Myc signalling pathway, promoting liver fibrosis and aerobic glycolysis in LX-2 cells and mice. It stabilises TRIM25 through post-translational modification, reduces its self-ubiquitination, and enhances the stability of c-Myc [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In vivo, FAK inhibitors block this pathway, alleviating liver fibrosis and aerobic glycolysis. These findings suggest that FAK inhibitors may be a potential therapeutic approach for liver fibrosis, and that TRIM25 is a key mediator in FAK-driven fibrotic progression.\u003c/p\u003e\u003cp\u003eIn summary, our study shows that FAK promotes liver fibrosis and aerobic glycolysis at cellular, animal, and tissue levels. Specific FAK inhibitors could mitigate these processes. FAK regulates the TRIM25/FBXW7/c-Myc pathway by binding to TRIM25\u0026rsquo;s RING, BOX, and SPRY regions, enhancing liver fibrosis and glycolysis. Despite limitations, such as using one hepatic stellate cell type, our findings lay the foundation for further exploration of liver fibrosis causes and management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Institutional Review Board of The Affiliated Hospital of Guizhou Medical University (2023 Lunshen 401). Written informed consent was obtained from all participants. All experiments, including those involving mice, complied with ethical regulations and were approved by the Animal Ethics Committee of Guizhou Medical University (No. 2403086).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author\u0026nbsp;upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Program of the Guizhou\u003c/p\u003e\n\u003cp\u003eProvince (No. [2021] 094.), the National Natural Science Foundation of China (No.82060116 and No.82260129), the Guizhou Provincial Science and Technology Program (QKH JC-ZK [2023]-214) and The Doctoral Research Start-up Fund Project of Guizhou Medical University Affiliated Hospital (No.gyfybsky[2021]-63 and No.gyfybsky-2025-10)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the conception and design of the study. Xue-Ke Zhao designed and supervised the study, and reviewed the final manuscript. Lu Han performed the majority of the experiments and drafted the initial manuscript. Fan Lu contributed to revising the manuscript. Gao-Liang Zou was responsible for conducting the WB experiments. Qing-Xiu Zhang, Ya Zhang, and Tao Ran conducted the animal experiments. Tao Huang performed statistical analysis. Hong-Fei Pu and Jing-Lin Wang collected the data. Jian-Chao Li assisted with experimental design. Hua-Yue Wu assisted with experimental procedures. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Bullet Edits Limited for the linguistic editing and proofreading of the manuscript.We would like to thank Guixiu Xie and Shuqiang Han for their support of the project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZubiete-Franco I, Fern\u0026aacute;ndez‐Tussy P, Barbier‐Torres L, Simon J, Fern\u0026aacute;ndez‐Ramos D, Lopitz‐Otsoa F, et al. Deregulated neddylation in liver fibrosis. Hepatology. 2017;65(2):694\u0026ndash;709.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOh Y, Park O, Swierczewska M, Hamilton JP, Park JS, Kim TH, et al. Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells. 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Dual focal adhesion kinase/Pyk2 inhibitor has positive effects on bone tumors: implications for bone metastases. Cancer. 2008;112(10):2313\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeitz HK, Bataller R, Cortez-Pinto H, Gao B, Gual A, Lackner C, et al. Alcoholic liver disease. Nat Rev Dis Primers. 2018;4(1):16.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Q, Li X, Cui K, Liu C, Wu M, Prochownik EV, et al. The MAP3K13-TRIM25-FBXW7alpha axis affects c-Myc protein stability and tumor development. Cell Death Differ. 2020;27(2):420\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQie S, Majumder M, Mackiewicz K, Howley BV, Peterson YK, Howe PH et al. Fbxo4-mediated degradation of Fxr1 suppresses tumorigenesis in head and neck squamous cell carcinoma. Nat Commun. 2017;8(1).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Focal adhesion kinase, tripartite motif protein 25, aerobic glycolysis, liver fibrosis, FAK inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-6993066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6993066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLiver fibrosis is marked by hepatic stellate cell (HSC) activation and increased glucose consumption. Focal adhesion kinase (FAK) upregulates c-Myc expression in HSCs, promoting aerobic glycolysis. This study explores how FAK promotes HSC activation and glycolysis via TRIM25. Immunohistochemistry (IHC) and Western blotting assessed the expression of FAK, TRIM25, FBXW7, c-Myc, and glycolysis-related proteins in human liver tissues and mouse models. Protein interactions were identified by co-immunoprecipitation (Co-IP) and LC-MS, and FAK and TRIM25 localization was observed by immunofluorescence. FAK inhibition reduced LX-2 cell activation, migration, and glycolysis. Co-IP and immunofluorescence confirmed FAK-TRIM25 interaction. FAK inhibits FBXW7-mediated c-Myc ubiquitination and enhances glycolysis by binding TRIM25\u0026rsquo;s RING, B-BOX, and SPRY regions. Inhibition of FAK improved liver fibrosis and glycolysis. FAK promotes HSC glycolysis through TRIM25 interaction, and its inhibition mitigates liver fibrosis, suggesting a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"FAK-TRIM25 Promotes HSC Activation and Glycolysis by Inhibiting c-Myc Ubiquitination via FBXW7","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-11 18:55:59","doi":"10.21203/rs.3.rs-6993066/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":"8c5615ac-eba6-4959-b2c8-eb4690b79fcc","owner":[],"postedDate":"July 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-30T00:34:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-11 18:55:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6993066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6993066","identity":"rs-6993066","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}