==== Front Genes Dis Genes Dis Genes & Diseases 2352-4820 2352-3042 Chongqing Medical University S2352-3042(22)00177-5 10.1016/j.gendis.2022.06.003 Rapid Communication Acute liver injury induces expression of FGF23 in hepatocytes via orphan nuclear receptor ERRγ signaling Jung Yoon Seok a1 Kim Yong-Hoon bc1 Radhakrishnan Kamalakannan ad1 Noh Jung-Ran b Choi Jung Hyeon b Kim Hyo-Jin a Jeong Jae-Ho e Dooley Steven f Lee Chul-Ho chullee@kribb.re.kr bc∗ Choi Hueng-Sik hsc@chonnam.ac.kr a∗∗ a School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea b Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea c Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea d Combinatorial Tumor Immunotherapy MRC, Chonnam National University Medical School, Hwasun-gun, Jeonnam 58128, Republic of Korea e Department of Microbiology, Chonnam National University Medical School, Gwangju 61186, Republic of Korea f Molecular Hepatology Section, Medical Faculty Mannheim, Heidelberg University, 69117 Heidelberg, Germany ∗ Corresponding author. School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of Korea. Fax: +82 62 530-0506.. chullee@kribb.re.kr ∗∗ Corresponding author. Fax: +82 62 530-0506. hsc@chonnam.ac.kr 1 These authors contributed equally to this work. 01 7 2022 5 2023 01 7 2022 10 3 679682 1 4 2022 5 5 2022 19 6 2022 © 2022 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. 2022 Chongqing Medical University https://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ==== Body pmcFibroblast growth factor 23 (FGF23) is an osteocyte- and osteoblast-derived hormone that primarily regulates phosphate and vitamin D metabolism. Circulatory FGF23 levels are abnormally increased in pathological conditions like acute or chronic kidney injury, resulting in disease progression as well as increased rates of morbidity and mortality.1 However, FGF23 production in acute liver injury is not fully investigated. In this study, we report that carbon tetrachloride (CCl4)-induced acute liver injury upregulates hepatic estrogen-related receptor gamma (ERRγ) and FGF23 gene expression and FGF23 secretion from liver. Hepatocyte specific depletion of ERRγ blunted CCl4-induced hepatic FGF23 promoter activity, FGF23 gene expression and FGF23 levels. Further, treatment with ERRγ-specific inverse agonist GSK5182 also efficiently inhibited CCl4-induced acute liver injury-mediated hepatic FGF23 gene expression and circulatory FGF23 levels in vivo. Taken together, these results firstly describe a detailed molecular mechanism of hepatic FGF23 gene expression induction in an acute liver injury condition. Further, we present evidence that inhibiting ERRγ transactivation by the small molecule GSK5182 may be a useful strategy to control the devastating circulatory levels of FGF23. A previous study suggested that FGF23 production is regulated by JAK/STAT signaling in mice liver.2 However, mechanism associated with FGF23 gene expression in liver disease are still unknown. In this context, we intraperitoneally injected WT mice with CCl4 (1 mL/kg body weight of 10% CCl4 dissolved in corn oil) for 6 h and assessed messenger RNA (mRNA) expression in major tissues including brain, heart, lung, liver, spleen, kidney and bone. ERRγ and FGF23 mRNA expressions were specifically increased in liver, but not in other major organs (Fig. 1A, B). To determine the expression pattern of CCl4-induced FGF23 gene expression, we injected mice with CCl4 for different time periods. Hematoxylin and eosin (H&E) staining of mouse liver samples showed that acute liver started to occur at the 3 h time point upon CCl4 injection, and kept worsening at 6, 12 and 24 h (Fig. S1A). FGF23 mRNA expression, protein expression and plasma intact FGF23 levels started to increase from 3 h on, reaching a peak after 6 h of CCl4 injection (Fig. S1B-D). Taken together, these results suggested that CCl4 induced FGF23 gene expression and secretion in mouse liver.Figure 1 CCl4-induced acute liver injury increases FGF23 gene expression and secretion in mouse liver through ERRγ.(A, B) Quantitative PCR analysis of total RNA obtained from the livers of mice injected with CCl4 (1 mL/kg body weight of 10% CCl4 dissolved in corn oil) for 6 h (n = 5 per group). (C–F) WT and ERRγ-LKO mice were injected with CCl4 for 6 h (n = 5 per groups). (C) Quantitative PCR analysis of total RNA isolated from livers. (D) Representative images of FGF23 immunohistochemical analysis in liver sections. (E) Representative in vivo images of hepatic FGF23 promoter WT-luciferase (Ad-FGF23-luc) activity in WT and ERRγ-LKO mice injected with or without CCl4 (n = 4 for WT-Con and ERRγ-LKO Con; n = 6 for WT-CCl4 and ERRγ-LKO CCl4 group). (F) Plasma FGF23 levels measured by ELISA. (G–I) WT mice were injected with CCl4 in the presence or the absence of GSK5182 and sacrificed after 6 h (n = 5 per group). (G) Quantitative PCR analysis of total RNA isolated from liver. (H) Representative images of FGF23 immunohistochemical analysis in liver sections. (I) Plasma FGF23 levels measured by ELISA. (J) Schematic diagram of ERRγ-mediated hepatic FGF23 gene expression and secretion in CCl4-induced acute liver injury. Data indicate mean ± SEM values. Data in (A) and (B) were analyzed by two-tailed Student's t test. Data in C, E, F, G and I were analyzed by ordinary one-way ANOVA with Tukey's multiple comparisons test. Significance levels denoted as ∗P < 0.05; ∗∗∗P < 0.001; n.s., not significant. Fig. 1 CCl4 injection possibly leads to nephrotoxicity and acute/chronic kidney injury results in increased production of renal FGF23. We investigated the gene expression in our model of CCl4-acute liver injury. The levels of pro-inflammatory cytokine interleukin-6 (IL6) mRNA were found to be increased in liver but not in kidney tissues in response to CCl4 injection (Fig. S2A). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, which are markers of liver injury, were significantly increased in the plasma of CCl4-injected mice (Fig. S2B, C). H&E staining of mouse liver and kidney samples showed that acute liver injury was induced by CCl4 injection without causing any damage to kidney (Fig. S2D). The kidney injury markers blood urea nitrogen (BUN) and creatinine remained unchanged between control and CCl4-injected groups (Fig. S2E, F). These results indicated that CCl4-acute liver injury increased FGF23 gene expression without causing renal damage. We previously reported that FGF23 gene expression is transcriptionally regulated in the liver by ERRγ in response to folic acid-induced acute kidney injury (FA-AKI).3 We also found that CCl4-acute liver injury resulted in increased hepatic ERRγ gene expression. To investigate whether ERRγ was the upstream regulator of hepatic FGF23 gene expression and secretion in our current study, we employed hepatocyte specific ERRγ knockout mice (ERRγ-LKO) and intraperitoneally injected with CCl4 for 6 h. The expression of hepatic ERRγ and FGF23 mRNA was increased in CCl4-injected WT mice, but was significantly inhibited in ERRγ-LKO mice (Fig. 1C). CCl4-mediated induction of hepatic FGF23 protein expression and plasma FGF23 levels was significantly blunted by hepatic loss of ERRγ expression (Fig. 1D, F). To elucidate the molecular mechanism underlying ERRγ-regulated FGF23 gene expression in response to CCl4-induced acute liver injury, we utilized mouse FGF23 promoter luciferase construct fused with adenovirus (Ad-FGF23-luc). WT and ERRγ-LKO mice were injected with Ad-FGF23-luc via tail vein and mice were treated 3 days post-injection with CCl4 for 6 h. In vivo imaging showed that hepatic FGF23 promoter activity was significantly increased in CCl4 injected mice compared with control mice. However, no difference was detected in hepatic FGF23 promoter activity between control and CCl4-injected ERRγ-LKO mice (Fig. 1E). This result showed that ERRγ directly binds with FGF23 promoter to transcriptionally regulate hepatic FGF23 gene expression in response to CCl4-acute liver injury. Then, we measured FGF23 levels in the plasma of WT and ERRγ-LKO mice treated with or without CCl4. Altogether, these results suggest that hepatic ERRγ expression was required to transcriptionally regulate hepatic FGF23 gene expression in CCl4-induced acute liver injury. Finally, we tested the pharmacological inhibition of ERRγ using inverse agonist GSK5182 to inhibit FGF23 gene expression in response to CCl4 injection. GSK5182 is an ERRγ-specific inverse agonist, which inhibits the transcriptional activity of ERRγ and thereby decreases the expression of ERRγ target genes. WT mice were injected with or without CCl4 in the presence or the absence of GSK5182. We found that GSK5182 treatment significantly inhibited CCl4-induced hepatic ERRγ and FGF23 mRNA expression in WT mice (Fig. 1G). Further, CCl4-induced hepatic FGF23 protein expression and plasma FGF23 levels were also inhibited by GSK5182 based on the results of immunohistochemical analysis and ELISA, respectively (Fig. 1H, I). These results suggest that inverse agonist-mediated inactivation of ERRγ transactivation significantly inhibits CCl4-mediated hepatic FGF23 gene expression. Hepatic FGF23 production was elevated in autosomal dominant polycystic kidney disease and childhood biliary atresia.4 We previously reported FA-AKI induced FGF23 gene expression and secretion by hepatocytes.3 AKI upregulated IL6 in kidney, which mediated organ-to-organ communication to induce haptic FGF23 production via ERRγ. Here, we showed that CCl4-acute liver injury upregulates hepatic FGF23 synthesis via ERRγ with significant increase in hepatic IL6 expression. Pro-inflammatory cytokine IL6 is the common factor in both FA-AKI and CCl4-acute liver injury and IL6 induces ERRγ gene expression in liver. In line with our data, Kumar et al recently reported upregulation of FGF23 production in total liver as response to CCl4 hepatotoxicity, diet-induced fatty liver, and bile-acid-induced cholestatic liver disease.5 They show in vitro that lipopolysaccharide mediated toll-like receptor (TLR) 4 signaling induced FGF23 production indirectly in macrophages, together with IL1β and TNFα in combination, and TLR2 agonist Pam2CSK3. The liver resident macrophages or Kupffer cells, but not the hepatocytes or hepatic stellate cells, were found as source of FGF23. They, however, did not dissect the cell types of FGF23 expression in the more general disease settings CCl4 treatment, high-fat diet feeding or bile duct ligation. In the current study, we report that hepatocytes are the major source of FGF23 in CCl4-induced acute liver injury and confirmed the finding by showing significantly decreased FGF23 production ERRγ-LKO mice. ERRγ is a key regulator of hepatic FGF23 production in vivo in response to CCl4-induced acute liver injury (Fig. 1J). Hepatocyte-specific ablation of ERRγ expression or inverse agonist-mediated inhibition of ERRγ transcriptional activity significantly blunted CCl4-induced hepatic FGF23 synthesis. Therefore, we suggest that blocking the ERRγ signaling is an attractive strategy to reduce pathologically abnormal circulatory FGF23 levels in acute liver injury. Ethics declaration All animal procedures were approved by the Institutional Animal Care and Use Committee of KRIBB (KRIBB-AEC-20135). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Author contributions Y.S.J., Y.-H.K., C.H.L., and H.-S.C. designed research. Y.S.J., Y.-H.K., H.-J.K., J.-R.N., and J.H.C. performed the experiments. Y.S.J., Y.-H.K., K.R., J.-H.J., and S.D. analyzed and interpreted the results. Y.S.J., Y.-H.K., K.R., and H.-S.C. drafted and revised the manuscript. All authors reviewed and agreed with the manuscript content. Conflict of interests Authors declare no conflict of interests. Funding This work was supported by the National Research Foundation (NRF) basic science research program Korean government (Ministry of Science and ICT), Republic of Korea (No. 2020R1A6A3A01096145, 2020) (No. NRF-2019R1C1C1005319, 2019) (No. NRF-2017R1A6A3A04006167, 2017) (No. NRF-2020R1A2C3006952, 2020); and (No. NRF-2021R1A2C3004923, 2021). The Federal Ministry of Education and Research-Liver Systems Medicine Program of the Stiftung für Biomedizinische Alkoholforschung, Germany (No. PTJ-031L0043). Appendix A Supplementary data The following are the Supplementary data to this article:Multimedia component 1 Multimedia component 1 Multimedia component 2 Multimedia component 2 Multimedia component 3 Multimedia component 3 Peer review under responsibility of Chongqing Medical University. Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.gendis.2022.06.003. ==== Refs References 1 Schnedl C. Fahrleitner-Pammer A. Pietschmann P. FGF23 in acute and chronic illness Dis Markers 2015 2015 358086 2 Daryadel A. Ruiz P.A. Gehring N. Systemic Jak 1 activation provokes hepatic inflammation and imbalanced FGF23 production and cleavage Faseb J 35 2 2021 e21302 3 Radhakrishnan K. Kim Y.H. Jung Y.S. Orphan nuclear receptor ERR-γ regulates hepatic FGF23 production in acute kidney injury Proc Natl Acad Sci U S A 118 16 2021 e2022841118 4 Wasserman H. Ikomi C. Hafberg E.T. Two case reports of FGF23-induced hypophosphatemia in childhood biliary atresia Pediatrics 138 2 2016 e20154453 5 Kumar P. Liu Y. Shen Y. Mouse liver injury induces hepatic macrophage FGF23 production PLoS One 17 3 2022 e0264743