Abstract
Dual-specificity phosphatase 6 (DUSP6) is a phosphatase specific for extracellular signal-regulated kinase (ERK). Dusp6-knockout mice are resistant to diet-induced hepatic steatosis, which appears to be linked to the downregulation of cytochrome P450 4 A (CYP4A); however, its mechanism remains unclear. This study aimed to elucidate how DUSP6 regulates CYP4A11 in human hepatocyte-lineage cells by focusing on forkhead box O1 (FOXO1). HepG2 and HuH-7 cells were challenged with palmitic acid and oleic acid to induce lipid accumulation while manipulating the expression of DUSP6, FOXO1, CYP4A11, ERK, and/or AKT. Lipid accumulation was reduced by DUSP6 knockdown, resulting in decreased CYP4A11 expression despite elevated phosphorylated ERK, AKT, and FOXO1. Inhibition of ERK increased lipid accumulation, while simultaneous inhibition of ERK and AKT decreased it. Knockdown of FOXO1 or induced expression of DUSP6 increased CYP4A11 expression and lipid accumulation, whereas induced expression of FOXO1 decreased them. Chromatin-immunoprecipitation showed that FOXO1 bound to CYP4A11 promoter. Immunoprecipitations revealed that DUSP6 bound to and anchored FOXO1 in the cytoplasm. These results indicate that DUSP6 interferes with FOXO1’s repressive activity towards CYP4A11 by sequestering it in the cytoplasm and preventing its nuclear translocation, which ultimately unleashes CYP4A11 and promotes lipid accumulation.
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The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
References
Lang, R., Hammer, M. & Mages, J. DUSP Meet immunology: dual specificity MAPK phosphatases in control of the inflammatory response. J. Immunol. 177, 7497–7504. https://doi.org/10.4049/jimmunol.177.11.7497 (2006).
Muda, M. et al. MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase. J. Biol. Chem. 271, 4319–4326 (1996).
Furukawa, T., Tanji, E., Xu, S. & Horii, A. Feedback regulation of DUSP6 transcription responding to MAPK1 via ETS2 in human cells. Biochem. Biophys. Res. Commun. 377, 317–320. https://doi.org/10.1016/j.bbrc.2008.10.003 (2008).
Furukawa, T., Sunamura, M., Motoi, F., Matsuno, S. & Horii, A. Potential tumor suppressive pathway involving DUSP6/MKP-3 in pancreatic cancer. Am. J. Pathol. 162, 1807–1815. https://doi.org/10.1016/S0002-9440(10)64315-5 (2003).
Chan, D. W. et al. Loss of MKP3 mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer cells. Carcinogenesis 29, 1742–1750. https://doi.org/10.1093/carcin/bgn167 (2008).
Okudela, K. et al. Down-regulation of DUSP6 expression in lung cancer: its mechanism and potential role in carcinogenesis. Am. J. Pathol. 175, 867–881. https://doi.org/10.2353/ajpath.2009.080489 (2009).
Wang, X. et al. Utility of patient-derived xenografts to evaluate drug sensitivity and select optimal treatments for individual non-small-cell lung cancer patients. Mol. Med. 30, 209. https://doi.org/10.1186/s10020-024-00934-4 (2024).
Png, C. W. et al. DUSP6 regulates Notch1 signalling in colorectal cancer. Nat. Commun. 15, 10087. https://doi.org/10.1038/s41467-024-54383-y (2024).
Feng, B. et al. Mitogen-activated protein kinase phosphatase 3 (MKP-3)-deficient mice are resistant to diet-induced obesity. Diabetes 63, 2924–2934. https://doi.org/10.2337/db14-0066 (2014).
Liu, R. et al. Mice lacking DUSP6/8 have enhanced ERK1/2 activity and resistance to diet-induced obesity. Biochem. Biophys. Res. Commun. 533, 17–22. https://doi.org/10.1016/j.bbrc.2020.08.106 (2020).
Pfuhlmann, K. et al. Dual specificity phosphatase 6 deficiency is associated with impaired systemic glucose tolerance and reversible weight retardation in mice. PLoS One. 12, e0183488. https://doi.org/10.1371/journal.pone.0183488 (2017).
Jiang, C. et al. Ablation of Dual-Specificity phosphatase 6 protects against nonalcoholic fatty liver disease via cytochrome P450 4A and Mitogen-Activated protein kinase. Am. J. Pathol. 193, 1988–2000. https://doi.org/10.1016/j.ajpath.2023.09.003 (2023).
Powell, P. K., Wolf, I. & Lasker, J. M. Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes. Arch. Biochem. Biophys. 335, 219–226. https://doi.org/10.1006/abbi.1996.0501 (1996).
Johnson, A. L., Edson, K. Z., Totah, R. A. & Rettie, A. E. Cytochrome P450 omega-Hydroxylases in inflammation and cancer. Adv. Pharmacol. 74, 223–262. https://doi.org/10.1016/bs.apha.2015.05.002 (2015).
Galili, N. et al. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat. Genet. 5, 230–235. https://doi.org/10.1038/ng1193-230 (1993).
Xing, Y. Q. et al. The regulation of FOXO1 and its role in disease progression. Life Sci. 193, 124–131. https://doi.org/10.1016/j.lfs.2017.11.030 (2018).
Behl, T. et al. Exploring the genetic conception of obesity via the dual role of FoxO. Int. J. Mol. Sci. 22 https://doi.org/10.3390/ijms22063179 (2021).
Li, T. et al. Forkhead box transcription factor O1 inhibits cholesterol 7alpha-hydroxylase in human hepatocytes and in high fat diet-fed mice. Biochim. Biophys. Acta. 1791, 991–996. https://doi.org/10.1016/j.bbalip.2009.05.004 (2009).
Tang, L. et al. FOXO1-regulated LncRNA CYP1B1-AS1 suppresses breast cancer cell proliferation by inhibiting neddylation. Breast Cancer Res. Treat. 202, 397–408. https://doi.org/10.1007/s10549-023-07090-z (2023).
Zhu, J. et al. Eicosatrienoic acid inhibits estradiol synthesis through the CD36/FOXO1/CYP19A1 signaling pathway to improve PCOS in mice. Biochem. Pharmacol. 229, 116517. https://doi.org/10.1016/j.bcp.2024.116517 (2024).
Konstandi, M., Cheng, J. & Gonzalez, F. J. Sex steroid hormones regulate constitutive expression of Cyp2e1 in female mouse liver. Am. J. Physiol. Endocrinol. Metab. 304, E1118–1128. https://doi.org/10.1152/ajpendo.00585.2012 (2013).
Duan, S. et al. Loss of FBXO31-mediated degradation of DUSP6 dysregulates ERK and PI3K-AKT signaling and promotes prostate tumorigenesis. Cell. Rep. 37, 109870. https://doi.org/10.1016/j.celrep.2021.109870 (2021).
Stulpinas, A. et al. Crosstalk between protein kinases AKT and ERK1/2 in human lung tumor-derived cell models. Front. Oncol. 12, 1045521. https://doi.org/10.3389/fonc.2022.1045521 (2022).
Dent, P. Crosstalk between ERK, AKT, and cell survival. Cancer Biol. Ther. 15, 245–246. https://doi.org/10.4161/cbt.27541 (2014).
Dumaz, N. & Marais, R. Raf phosphorylation: one step forward and two steps back. Mol. Cell. 17, 164–166. https://doi.org/10.1016/j.molcel.2005.01.001 (2005).
Wu, Z. et al. MAPK phosphatase-3 promotes hepatic gluconeogenesis through dephosphorylation of forkhead box O1 in mice. J. Clin. Invest. 120, 3901–3911. https://doi.org/10.1172/JCI43250 (2010).
Matsuzaki, H., Daitoku, H., Hatta, M., Tanaka, K. & Fukamizu, A. Insulin-induced phosphorylation of FKHR (Foxo1) targets to proteasomal degradation. Proc. Natl. Acad. Sci. U S A. 100, 11285–11290. https://doi.org/10.1073/pnas.1934283100 (2003).
Huang, H. et al. Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation. Proc. Natl. Acad. Sci. U S A. 102, 1649–1654. https://doi.org/10.1073/pnas.0406789102 (2005).
Guo, S. et al. Phosphorylation of Serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J. Biol. Chem. 274, 17184–17192. https://doi.org/10.1074/jbc.274.24.17184 (1999).
Basaranoglu, M., Basaranoglu, G. & Senturk, H. From fatty liver to fibrosis: a Tale of second hit. World J. Gastroenterol. 19, 1158–1165. https://doi.org/10.3748/wjg.v19.i8.1158 (2013).
Robertson, G., Leclercq, I. & Farrell, G. C. Nonalcoholic steatosis and steatohepatitis. II. Cytochrome P-450 enzymes and oxidative stress. Am. J. Physiol. Gastrointest. Liver Physiol. 281, G1135–1139. https://doi.org/10.1152/ajpgi.2001.281.5.G1135 (2001).
Arif Asghar, M. et al. NRF2 pathway activation as a molecular toxicology mechanism in oxidative stress and lipid metabolic disorders. J. Biochem. Mol. Toxicol. 39, e70633. https://doi.org/10.1002/jbt.70633 (2025).
Kohjima, M. et al. Re-evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease. Int. J. Mol. Med. 20, 351–358 (2007).
Gao, H., Cao, Y., Xia, H., Zhu, X. & Jin, Y. CYP4A11 is involved in the development of nonalcoholic fatty liver disease via ROS–induced lipid peroxidation and inflammation. Int. J. Mol. Med. 45, 1121–1129. https://doi.org/10.3892/ijmm.2020.4479 (2020).
Ni, K. D. & Liu, J. Y. The functions of cytochrome P450 omega-hydroxylases and the associated eicosanoids in Inflammation-Related diseases. Front. Pharmacol. 12, 716801. https://doi.org/10.3389/fphar.2021.716801 (2021).
Hoopes, S. L., Garcia, V., Edin, M. L., Schwartzman, M. L. & Zeldin, D. C. Vascular actions of 20-HETE. Prostaglandins Other Lipid Mediat. 120, 9–16. https://doi.org/10.1016/j.prostaglandins.2015.03.002 (2015).
Tsai, I. J., Croft, K. D., Puddey, I. B., Beilin, L. J. & Barden, A. 20-Hydroxyeicosatetraenoic acid synthesis is increased in human neutrophils and platelets by angiotensin II and endothelin-1. Am. J. Physiol. Heart Circ. Physiol. 300, H1194–1200. https://doi.org/10.1152/ajpheart.00733.2010 (2011).
Hardwick, J. P. Cytochrome P450 Omega hydroxylase (CYP4) function in fatty acid metabolism and metabolic diseases. Biochem. Pharmacol. 75, 2263–2275. https://doi.org/10.1016/j.bcp.2008.03.004 (2008).
Cheng, J. et al. FOXO1 induced fatty acid oxidation in hepatic cells by targeting ALDH1L2. J. Gastroenterol. Hepatol. 39, 2197–2207. https://doi.org/10.1111/jgh.16662 (2024).
Guo, L. et al. Similarities and differences in the expression of drug-metabolizing enzymes between human hepatic cell lines and primary human hepatocytes. Drug Metab. Dispos. 39, 528–538. https://doi.org/10.1124/dmd.110.035873 (2011).
Arzumanian, V., Pyatnitskiy, M. & Poverennaya, E. Comparative transcriptomic analysis of three common liver cell lines. Int. J. Mol. Sci. 24 https://doi.org/10.3390/ijms24108791 (2023).
Huggett, Z. J. et al. A comparison of primary human hepatocytes and hepatoma cell lines to model the effects of fatty Acids, Fructose and glucose on liver cell lipid accumulation. Nutrients 15 https://doi.org/10.3390/nu15010040 (2022).
Arzumanian, V. A., Timofeeva, E. V., Kiseleva, O. I. & Poverennaya, E. V. Drug-Metabolizing gene expression identity: comparison across liver tissues and model cell lines. Biomedicines 13 https://doi.org/10.3390/biomedicines13112722 (2025).
Yang, L. et al. RING finger protein 4 (RNF4) reduces nonalcoholic fatty liver disease accumulation by promoting the sumoylation of HIF-2alpha and regulating the PPARalpha signaling pathway. IUBMB Life. 77, e70047. https://doi.org/10.1002/iub.70047 (2025).
Wang, Y. et al. The ErChen Decoction and its active compounds ameliorate Non-Alcoholic fatty liver disease through activation of the AMPK signaling pathway. Pharmaceuticals (Basel). 18 https://doi.org/10.3390/ph18111707 (2025).
Schicht, G., Seidemann, L., Haensel, R., Seehofer, D. & Damm, G. Critical investigation of the usability of hepatoma cell lines HepG2 and Huh7 as models for the metabolic representation of resectable hepatocellular carcinoma. Cancers (Basel). 14. https://doi.org/10.3390/cancers14174227 (2022).
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This work was supported by JSPS KAKENHI Grant Number JP23K06477 to YS.
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Masanobu Kimura: Investigation, Formal analysis, Writing-Original draft preparation. Yuriko Saiki: Conceptualization, Methodology, Investigation, Visualization, Funding acquisition, Writing - Review & Editing. Kosei Iwata: Investigation. Keigo Murakami: Investigation. Toru Furukawa: Conceptualization, Methodology, Visualization, Supervision, Writing - Review & Editing. All authors read and approved the final manuscript.
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Kimura, M., Saiki, Y., Iwata, K. et al. Dual-specificity phosphatase 6 interferes with the repressive activity of forkhead box O1 towards CYP4A11 that mediates lipid accumulation in the liver. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35118-z
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DOI: https://doi.org/10.1038/s41598-026-35118-z
