A Low-Molecular-Weight Compound K7174 Represses Hepcidin: Possible Therapeutic Strategy against Anemia of Chronic Disease

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of the hepcidin gene, HAMP, is increased in patients with anemia of chronic disease. Previously, the synthetic compound K7174 was identified through chemical screening as a novel inhibitor of the adhesion of monocytes to cytokine-stimulated endothelial cells. K7174 also ameliorated anemia induced by inflammatory cytokines in mice, which suggests a possible involvement of hepcidin regulation. The present study was performed to assess the impact of K7174 on hepcidin expression in a human hematoma cell line and in mice in vivo. We first demonstrated that K7174 treatment in HepG2 cells significantly decreased HAMP expression. Then, we conducted microarray analysis to determine the molecular mechanism by which K7174 inhibits HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Surprisingly, we found that K7174 strongly induced GDF15, known as a negative regulator of HAMP expression. Western blotting analysis as well as ELISA confirmed the induction of GDF15 by K7174 treatment. Furthermore, K7174-mediated HAMP suppression was rescued by the silencing of GDF15 expression. Interestingly, we found that K7174 also upregulates CEBPB. Promoter analysis and chromatin immunoprecipitation analysis revealed that CEBPB could contribute to K7174-mediated transcriptional activation of GDF15. Subsequently, we also examined whether K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver samples from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp expression, as compared to control mice. Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice. In conclusion, K7174 inhibits hepcidin expression partly by inducing GDF15. K-7174 may be a potential therapeutic option to treat anemia of chronic disease.铁调素（Hepcidin）是机体主要的铁调节激素，可调控全身性铁吸收及细胞内储存铁的再动员。慢性疾病贫血患者体内铁调素基因HAMP的表达水平显著升高。此前，研究人员通过化学筛选发现合成化合物K7174是单核细胞与细胞因子刺激的内皮细胞黏附的新型抑制剂。K7174还可改善小鼠体内炎症细胞因子诱导的贫血，这提示其可能参与铁调素的调控过程。本研究旨在评估K7174对人血肿细胞系及活体小鼠中铁调素表达的影响。 我们首先证实，在HepG2细胞中经K7174处理后，HAMP的表达显著下调。随后，我们通过微阵列分析（microarray analysis）探究K7174抑制HAMP表达的分子机制。转录组分析（Transcriptional profiling）证实了HAMP的下调。令人意外的是，我们发现K7174可强力诱导生长分化因子15（GDF15）的表达，而GDF15是已知的HAMP表达负调控因子。蛋白质免疫印迹（Western blotting）分析及酶联免疫吸附测定（ELISA）均证实，K7174处理可诱导GDF15的表达。此外，沉默GDF15的表达可挽救K7174介导的HAMP表达抑制。 值得注意的是，我们发现K7174还可上调CEBPB的表达。启动子分析及染色质免疫沉淀分析（chromatin immunoprecipitation analysis）显示，CEBPB可参与K7174介导的GDF15转录激活。随后，我们还检测了K7174对小鼠体内铁调素表达的抑制作用。对K7174处理小鼠的肝脏样本进行定量实时逆转录聚合酶链式反应（Quantitative RT-PCR，qRT-PCR）分析结果显示，与对照组小鼠相比，处理组小鼠体内Gdf15的表达显著上调，而Hamp的表达显著下调。此外，K7174处理小鼠的血清铁调素浓度也显著降低。 综上，K7174可通过诱导GDF15的表达部分抑制铁调素的表达。K7174有望成为慢性疾病贫血的潜在治疗选择。