A Low-Molecular-Weight Compound K7174 Represses Hepcidin: Possible Therapeutic Strategy Against Anemia Of Chronic Disease
Authors: Fujiwara Tohru , Ikeda Takashi , Nagasaka Yuki , Okitsu Yoko , Fukuhara Noriko , Onishi Yasushi , Ishizawa Kenichi , Ichinohasama Ryo , Tomosugi Naohisa , Harigae Hideo 
Published: October 2019

Abstract

Background
Hepcidin (HAMP) is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of HAMP increased in patients with anemia of chronic disease. Previously, a synthesized compound K7174 (Kowa Company Ltd., Tokyo, Japan) was identified through chemical screening as a novel inhibitor for the adhision of monocytes to cytokine-stimulated endothelial cells (Umetani et al. BBRC 2000). Interstingly, K7174 restored anemia induced by inflammatory cytokines in mice (Imagawa et al. FASEB J 2003), implying that K7174 might modulate hepcidin level. In the present study, we assessed the impact of K7174 on hepcidin expression based on human hematoma cell line and in vivo mice.

Method
The HepG2 hematoma cells as well as K562 erythroid cells were used for the analyses. The cells were treated with K7174 at doses of 10 and 20 uM for 24 h. For transcription profiling, Human Oligo chip 25K (Toray) were used for K7174-treated HepG2 cells. Western blotting and quantitative chromatin immunoprecipitation (ChIP) analyses were performed with antibodies to GDF15 (abcam), C/EBPbeta (CEBPB) (C-19) (Santa Cruz), Smad1 and Phospho-Smad1-5-8 (CST). For GDF15 knockdown in HepG2 cells, anti-GDF15 siRNA (Thermo Scientific Dharmacon) and Lipofectamine RNAiMAX (Invitrogen) were used. GDF15 promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Human GDF15 concentration in the K7174-treated media was evaluated with ELISA (R&D systems). For in vivo analysis, ICR mice were injected intraperitoneally with PBS (control) or 30 mg/kg K-7174, respectively, days 0, 1, 2, 3, 5, 6, 7 and 8, and the samples were taken on day 9. Serum hepcidin1 concentration was determined with LC-MS/MS method (MCProt Biotechnology, Kanazawa, Japan).

Results
We first demonstrated that K7174 treatment (20 uM) in HepG2 cells significantly decreased HAMP expression (> 2-fold). Thus, we next conducted microarray analysis to reveal the molecular mechanism by which K7174 inhibits the HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Interestingly, K7174 strongly induced GDF15 (10-fold), a negative regulator of HAMP expression (Tanno et al. Nat Med 2007). Quantitative RT-PCR, Western blotting as well as ELISA analyses confirmed the induction of GDF15 by K7174 treatment. Furthermore, siRNA-mediated GDF15 knockdown during K7174 treatment significantly re-activated HAMP expression, suggesting that the increase of GDF15 induced by K7174 was responsible for the HAMP downregulation. Noticeably, we also found that K7174 upregulates CEBPB (2.9-fold). Promoter assay and quantitative ChIP analysis demonstrated that K7174-mediated upregulation of CEBPB contributes to the transcriptional activation of GDF15. On the other hand, the microarray analysis and quantitative RT-PCR-based validation identified the significant K7174-mediated downregulation of BMP4 (2.6-fold), a positive regulator of HAMP expression (Babitt et al. Nat Genet 2006). However, the level of SMAD1-5-8 phosphorylation in K7174-treated cells remained unchanged, implying that BMP-SMAD signaling might not be essential in K7174-mediated HAMP suppression.
Next, we assessed if K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver sample from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp (n = 8, p< 0.05). Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice (Average: 138.1 and 110.4 ng/mL for K7174-treated and control mice, respectively. n = 8, p< 0.05). Beyond the regulation of Gdf15 in hepatocytes, erythroid cells have also been suggested to be one of the main sources of GDF15. Thus, we treated K7174 with K562 erythroid cells, and confirmed significant increase of GDF15, suggesting that systemic administration of K7174 may act on hepatocytes as well as erythroid cells to stimulate GDF15 production.

Conclusion
Our in vitro and in vivo analyses suggested that K7174 suppresses HAMP expression through modulating GDF15 expression. K7174 may be considered as a potential therapeutic option to treat anemia of chronic disease.

Disclosures:
No relevant conflicts of interest to declare.