Hepcidin encoded by the HAMP gene is known to be responsible for iron homeostasis in various mammals (1). It has been demonstrated that targeted disruption of hepcidin in the liver can reiterate the hemochromatosis phenotype accompanied by increased plasma iron and massive parenchymal iron accumulation (2). Hepcidin-deficient mouse models appear to be resistant to tissue damage and dysfunction during pulmonary iron overload with normal expression of upstream stimulatory factor 2 (USF2) (3, 4). Ceruloplasmin known to be involved in iron metabolism is deregulated in hepcidin-deficient mice (5). Conversely, mouse models of hepcidin transgenic overexpression in the liver have demonstrated severe iron-deficiency anemia (6). Hepcidin can also prevent iron overload and improve erythropoiesis in a mouse model of β-thalassemia (7). Hepatic hepcidin production is increased due to infection, inflammation, and severe iron overload, whereas it is decreased by hypoxia, iron-deficiency anemia, erythropoietin, and growth factors (8). Our recent study has shown that b-cell translocation gene 2 (BTG2)-yin yang 1 (YY1)-hepcidin signaling network controls mammalian iron homeostasis in the liver (8). The aim of the present study was to aimed to identify novel pathways associated with iron metabolism and gluconeogenesis.
Cereblon (CRBN) is a 442-amino acid protein exhibiting multiple functions in human brains and other tissues. It is associated with memory and learning (9). CRBN is also as an important molecular target of thalidomide (an antiemetic agent)-mediated teratogenicity (10). CRBN is predominantly expressed in the brain and moderately expressed in diverse tissues. It is a component of an E3 ubiquitin ligase complex by interacting with damage-specific DNA-binding protein 1 (DDB1), cullin 4 (Cul4), regulator of cullins 1 (ROC1), and ring box protein 1 (RBX1). Moreover, CRBN can directly interact with large-conductance calcium-activated potassium channels (BKCa), voltage-gated chloride channel (ClC)-2, ikaros zinc finger (IKZF)1, IKZF3, and AMP‐activated protein kinase (AMPK) (9-11). It plays a vital role in the regulation of ion transport and negatively modulates the AMPK signaling pathway
Kruppel-like factor 15 (KLF15) is a member of the KLF transcription factor family. It contains a zinc finger DNA-binding domain. KLF15 is expressed in diverse tissues such as the liver, kidney, pancreas, muscle, and heart (17). It is upregulated by glucagon and glucocorticoids during starvation or under diabetic conditions but downregulated by feeding and insulin (18, 19). KLFs are also involved in fibrosis, obesity, cardiovascular disease, cancer, and inflammatory conditions. KLF15 is a key positive regulator of gluconeogenesis but a negative regulator of cardiac hypertrophy and fibrosis (18, 20). However, the critical role of KLF15 in the regulation of iron metabolism through hepcidin gene expression remains unexplored.
In this study, we demonstrated that fasting and FSK treatment could significantly elevate hepatic hepcidin gene expression and production by increasing the expression of CRBN and KLF15. Moreover, disruption of CRBN and KLF15 markedly decreased hepcidin gene expression, eventually decreasing hepcidin secretion under FSK treatment. Our findings suggest that CRBN and KLF15 are mediators of fasting-induced hepatic hepcidin expression and its biosynthesis. Therefore, targeting CRBN and KLF15 might be a therapeutically important strategy to combat metabolic dysfunction.
Gluconeogenic stimuli are known to regulate iron metabolism through hepcidin induction in starved mice (21). Cereblon (CRBN) deficiency is known to prevent diet-induced obesity and insulin resistance by increasing AMPK activation (13). We analyzed the expression of genes in livers of fasted mice to explore the link of
We investigated the crucial role of
To understand effects of KLF15 on hepcidin gene expression and production, we modulated hepatic hepcidin gene expression using an adenoviral delivery system in primary mouse hepatocytes. As shown in Fig. 3A,
Next, we explored whether KLF15 could directly control the transcriptional activity of hepcidin induced by FSK treatment. We observed that FSK treatment or transiently expressed
Previous studies have reported that gluconeogenic signals can modulate hepatic hepcidin gene expression and its biosynthesis in mice (21, 23, 24). Hepcidin is an established modulator of iron metabolism, inflammation, hypoxia, and erythropoiesis (25, 26). During fasting conditions, the BTG2-KLF15 signaling network can modulate the biosynthesis of hepatic fibroblast growth factor 21 (17). Our previous study has shown that AMPK is negatively regulated by CRBN in mice with alcoholic liver disease (16). However, there is no information regarding the potential role of CRBN in modulating hepatic hepcidin gene expression or its secretion. Our current results demonstrated that elevated CRBN expression by the fasting state or FSK treatment could control the production of hepatic hepcidin by promoting
Glucagon and glucocorticoids are known to induce the transcription factor KLF15 during a fasting state, whereas insulin is known to downregulate KLF15 expression under a fed condition (18-20). It is known that KLF15 can modulate fibrosis, obesity, cardiovascular disease, cancer, and inflammatory conditions. Based on this information, we further characterized the novel molecular mechanisms underlying the fasting-mediated induction of hepcidin gene transcription by the CRBN-KLF15 signaling pathway in hepatocytes. As depicted in Fig. 3, KLF15 positively regulated hepcidin gene expression and production in primary mouse hepatocytes during FSK treatment. For the first time, we identified a KLF15-binding site on the
Hepcidin is produced due to iron loading and inflammation at a high rate in the liver. It is cleared from the circulation by kidneys to maintain iron balance in the body (1, 7, 27). Hepcidin deficiency can result in iron overload (3-5, 28), whereas overloading of hepcidin can lead to the development of iron-restrictive anemia, autoimmune inflammatory disorders, chronic kidney disease, cancers, infections, and inherited iron-refractory iron-deficiency anemia (25, 27, 28). Psychological stress and overload exercise can reduce serum iron levels and result in the inhibition of erythropoiesis in rats (29, 30). All these studies have shown that short-term food deprivation and reduced iron levels could lead to psychological stress and depression symptoms in patients with anorexia or other eating disorders. Our results indicate that a fasting condition can increase hepcidin production but decrease iron levels. Interestingly, hepcidin treatment strikingly reduced serum iron levels in mice (Fig. 1), indicating an inverse correlation between hepcidin and serum iron levels under a fasting condition. In addition, iron-mediated hepcidin regulation is associated with the bone morphogenetic protein (BMP)-SMAD pathway, whereas inflammation-mediated regulation is associated with both the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription signaling axis and the BMP-SMAD pathway (1, 7, 25, 26, 31). Increased expression of hepcidin regulation under a fasting state can lead to decreased serum iron levels via the upregulation of CRBN, KLF15, and HAMP. Therefore, altering the serum hepcidin rate can upregulate its circulating concentration in a fasting state, consequently changing serum iron flow.
In conclusion, our current study demonstrates that hepcidin is a possible novel target of CRBN and that CRBN encourages hepatic hepcidin gene expression and production by inducing KLF15 expression when exposed to gluconeogenic signals such as a fasting state or FSK treatment. We speculate that the increase in CRBN expression induced by gluconeogenic stimuli can efficiently modulate hepatic hepcidin metabolism by enhancing the expression of KLF15. Furthermore, upregulated CRBN can decrease serum iron levels under a fasting state (Fig. 4D). This novel molecular mechanism involving hepatic hepcidin metabolism by the CRBN-KLF15 signaling network might provide a better understanding to develop potential therapeutic agents and important strategies to intervene metabolic dysfunction caused by iron-deficiency anemia.
Additional detailed methods are described in Supplementary information.
This research was supported by Kyungpook National University Development Project Research Fund, 2018 (to Y.D.K.).
The authors have no conflicting interests.