Hepcidin and iron regulatory proteins coordinately regulate ferroportin 1 expression in the brain of mice.

Iron plays an essential role in various cellular metabolic processes of the body. Maintenance of cellular iron homeostasis is particularly important for keeping the normal functions of the cells. Ferroportin 1 (FPN1) is the currently only known iron exporter on the cell membrane. It has been indicated that the regulation of FPN1 in response to the alteration of iron level mainly involves two processes, posttranscriptional repression by iron regulatory proteins (IRPs) and posttranslational degradation by hepcidin, the major iron-sensing hormone. However, whether there is any communication between the two types of regulations or which one plays dominant role has not been reported. In our study with IRP2-/- mice, we found that knockout of IRP2 increased FPN1 expression in the cerebral cortex of IRP2-/- mice, whereas the upregulation of FPN1 was more significant in IRP1/IRP2 dual knockdown fibroblasts. Interestingly, we found that the knockout of IRP2 severely affected the regulation effect of hepcidin on FPN1 in mouse brain. FPN1 level decreased dramatically in the brain of wild-type mice injected with hepcidin, but it did not decrease much in IRP2 knockout mice. Further investigation disclosed that the compromised hepcidin-FPN1 regulation in IRP2-/- cells was directly dependent on the existence of iron-responsive element (IRE) in FPN1 messenger RNA. These results indicate that IRPs and hepcidin coordinately regulate the FPN1 level in mice. This study will provide a more comprehensive understanding of the regulatory mechanisms of FPN1 expression.

The α1-adrenergic receptor is involved in hepcidin upregulation induced by adrenaline and norepinephrine via the STAT3 pathway.

Elevated body iron stores are associated with hypertension progression, while hypertension is associated with elevated plasma catecholamine levels in patients. However, there is a gap in our understanding of the connection between catecholamines and iron regulation. Hepcidin is a key iron-regulatory hormone, which maintains body iron balance. In the present study, we investigated the effects of adrenaline (AD) and norepinephrine (NE) on hepatic hepcidin regulation. Mice were treated with AD, NE, phenylephrine (PE, α1-adrenergic receptor agonist), prazosin (PZ, α1-adrenergic receptor antagonist), and/or propranolol (Pro, ß-adrenergic receptor antagonist). The levels of hepcidin, as well as signal transducer and activator of transcription 3 (STAT3), ferroportin 1 (FPN1), and ferritin-light (Ft-L) protein in the liver or spleen, were assessed. Six hours after AD, NE, or PE treatment, hepatic hepcidin mRNA levels increased. Pretreatment with PZ, but not Pro, abolished the effects of AD or NE on STAT3 phosphorylation and hepatic hepcidin expression. When mice were treated with AD or NE continuously for 7 days, an increase in hepatic hepcidin mRNA levels and serum hepcidin concentration was also observed. Meanwhile, the expected downstream effects of elevated hepcidin, namely decreased FPN1 expression and increased Ft-L protein and non-heme iron concentrations in the spleen, were observed after the continuous AD or NE treatments. Taken together, we found that AD or NE increase hepatic hepcidin expression via the α1-adrenergic receptor and STAT3 pathways in mice. The elevated hepatic hepcidin decreased FPN1 levels in the spleen, likely causing the increased iron accumulation in the spleen.

Nasal delivery of nanoliposome-encapsulated ferric ammonium citrate can increase the iron content of rat brain.

BACKGROUND: Iron deficiency in children can have significant neurological consequences, and iron supplementation is an effective treatment of choice. However, traditional routes of iron supplementation do not allow efficient iron delivery to the brain due to the presence of the blood-brain barrier. So an easily delivered iron formulation with high absorption efficiency potentially could find widespread application in iron deficient infants. RESULTS: In this study, we have developed and characterized a nanovesicular formulation of ferric ammonium citrate (ferric ammonium citrate nanoliposomes, FAC-LIP) and have shown that it can increase brain iron levels in rats following nasal administration. FAC was incorporated into liposomes with high efficiency (97%) and the liposomes were small (40 nm) and stable. Following intranasal delivery in rats, FAC-LIP significantly increased the iron content in the olfactory bulb, cerebral cortex, striatum, cerebellum and hippocampus, and was more efficient at doing so than FAC alone. No signs of apoptosis or abnormal cell morphology were observed in the brain following FAC-LIP administration, and there were no significant changes in the levels of SOD and MDA, except in the cerebellum and hippocampus. No obvious morphological changes were observed in lung epithelial cells or tracheal mucosa after nasal delivery, suggesting that the formulation was not overtly toxic. CONCLUSIONS: In this study, nanoscale FAC-LIP proved an effective system delivering iron to the brain, with high encapsulation efficiency and low toxicity in rats. Our studies provide the foundation for more detailed investigations into the applications of niosomal nasal delivery of liposomal formulations of iron as a simple and safe therapy for iron deficiency anemia. Graphical abstract The diagrammatic sketch of "Nasal delivery of nanoliposome-encapsulated ferric ammonium citrate can increase the iron content of rat brain". Nanoliposome-encapsulated ferric ammonium citrate (FAC-LIP) was successfully prepared and intranasal administration of FAC-LIP increased both the total iron contents and iron storage protein (FTL) expression in rat olfactory bulb, cerebral cortex, striatum and hippocampus, compared with those of FAC groups. Moreover, there was not overtly toxic affects to brain, lung epithelial cells and tracheal mucosa.

Astrocyte-derived hepcidin controls iron traffic at the blood-brain-barrier via regulating ferroportin 1 of microvascular endothelial cells.

Brain iron dysregulation associated with aging is closely related to motor and cognitive impairments in neurodegenerative diseases. The regulation of iron traffic at the blood-brain barrier (BBB) is crucial to maintain brain iron homeostasis. However, the specific mechanism has not been clarified in detail. Using various conditional gene knockout and overexpression mice, as well as cell co-culture of astrocyte and bEND.3 in the transwell, we found that astrocyte hepcidin knockdown increased the expression of ferroportin 1 (FPN1) of brain microvascular endothelial cells (BMVECs), and that it also induced brain iron overload and cognitive decline in mice. Moreover, BMVECs FPN1 knockout decreased iron contents in the cortex and hippocampus. Furthermore, hepcidin regulates the level of FPN1 of BMVECs with conditional gene overexpression in vivo and in vitro. Our results revealed that astrocytes responded to the intracellular high iron level and increased the secretion of hepcidin, which in turn diminished iron uptake at BBB from circulation through directly regulating FPN1 of BMVECs. Our results demonstrate that FPN1 of BMVECs is a gateway for iron transport into the brain from circulation, and the controller of this gateway is hepcidin secreted by astrocyte at its endfeet through physical contact with BMVECs. This regulation is indeed the major checkpoint for iron transport from the blood circulation to the brain. This study delineates the pathway and regulation of iron entry into the brain, providing potential therapeutic targets for iron dysregulation-related neurological diseases.

AnnexinA5 Might Suppress the Phenotype of Human Gastric Cancer Cells via ERK Pathway.

Gastric cancer is one of the most fatal diseases around the world. However, the mechanism of the development of gastric cancer is still not clarified. In addition, the anticancer drugs have cytotoxicity with different degrees. AnnexinA5, a member of the annexin family, has a great binding ability with the membrane phospholipid in a calcium dependent manner and is involved in the development of various cancers. This study aims to explore the influence of annexinA5 on human gastric cancer cells and whether it has the potential to be an auxiliary treatment to gastric cancer. In this study, the role of annexinA5 was detected from both the endogenous and the exogenous aspects on the gastric cancer cell lines MGC-803 and MKN-45. The cells were divided into a knockdown group in which RNA interference technique was used to suppress annexinA5 expression and a protein-supplementing group in which annexinA5 protein was added in the culture supernatant. After the suppression ratio of RNA interference was determined and the IC50 of annexinA5 protein was decided respectively, the cells' proliferation was detected by MTT assay, colony formation assay, and the expression of PCNA. FCM assay and PI staining methods were applied to test cell apoptosis and necrosis. To investigate whether ANXA5 influence cell metastasis, wound healing assay and transwell assay were employed. To further detect the mechanism of annexinA5 action, the signal pathway was examined with Western Blot method. When ANXA5 gene was knocked down, cell proliferation and metastasis were promoted, while cell apoptosis was suppressed. On the other hand, after the annexinA5 protein was applied to the gastric cancer cells, cell proliferation and metastasis were inhibited, while cell apoptosis and necrosis were promoted. AnnexinA5 played its role via ERK signal pathway. ANXA5 acted as tumor suppressor gene in the gastric cancer by suppressing ERK signal pathway and has the potentiality to be an auxiliary anticancer agent.

Iron overload induced by IRP2 gene knockout aggravates symptoms of Parkinson's disease.

Parkinson's disease (PD) is accompanied by iron overload in the brain. However, whether iron accumulation is the cause or effect of PD is still unknown. Iron regulatory protein 2 (IRP2) plays a critical role in keeping iron homeostasis, and our previous data showed that the deletion of the IRP2 gene caused iron deposits in organs of mice. Therefore, we further investigated the role of iron overload induced by IRP2 gene deletion in the development of the MPTP-induced PD mouse model in vivo, and the underlying regulatory mechanisms in primary cultures of astrocytes in vitro. Data from neurobehavioral, immunohistochemistry, TUNEL and Elisa studies showed that MPTP treatment enhanced the symptoms of PD in vivo, increased cell apoptosis and decreased dopamine levels in IRP2-/- mice. In addition, the expression of L-ferritin and iron contents increased significantly in the substantia nigra (SN) of IRP2-/- mice. Moreover, MPTP treatment significantly increased the expression of DMT1 (-IRE) and decreased the expression of TfR1 in IRP2-/- mice. Further investigations with primary cultures of astrocytes from IRP2-/- mice showed that MPP+ increased the expression of L-ferritin and DMT1 (-IRE), and decreased the expression of TfR1. Our results demonstrated that IRP2 gene deletion induced iron accumulation in the SN, which exacerbated the neuronal apoptosis and Parkinsonism symptoms. At the same time, IRP2 gene deletion increased the iron contents in astrocytes around neurons, which further decreased their protection for neurons and increased the cell apoptosis, ultimately forming a vicious cycle that leads to the onset and progression of PD.

Annexin A5 overexpression might suppress proliferation and metastasis of human uterine cervical carcinoma cells.

BACKGROUND: Annexin A5 (ANXA5) is a kind of Ca2+-dependent phospholipid binding protein which is involved in cell membrane dynamics and organization. Recent data showed that ANXA5 might involve in tumorigenesis. OBJECTIVE: To explore what role ANXA5 play in human uterine cervical carcinoma. MATERIALS AND METHODS: In this study, a recombined ANXA5 plasmid was constructed and uterine cervical carcinoma cell lines HeLa and SiHa were transfected with it. After ANXA5 overexpression was determined by Western Blot, cell proliferation test was detected by MTT assay and colony formation assay respectively. FACS assay and Hochest33258 staining methods were employed to detect cell apoptosis. To further investigate whether ANXA5 influence cell migration and invasion, wound healing assay and transwell assay were applied. At the same time, the relative mechanism was investigated. RESULTS: When ANXA5 expression increased, cell proliferation was inhibited by regulating the expression of bcl-2 and bax while cell metastasis was suppressed by regulating E-cadherin and MMP-9 expression. CONCLUSION: ANXA5 overexpression in the uterine cervical carcinoma might play important roles in cell proliferation and metastasis of uterine cervical cancer cells and act as an anti-cancer gene in uterine cervical cancer.

Astrocyte hepcidin is a key factor in LPS-induced neuronal apoptosis.

Inflammatory responses involving microglia and astrocytes contribute to the pathogenesis of neurodegenerative diseases (NDs). In addition, inflammation is tightly linked to iron metabolism dysregulation. However, it is not clear whether the brain inflammation-induced iron metabolism dysregulation contributes to the NDs pathogenesis. Herein, we demonstrate that the expression of the systemic iron regulatory hormone, hepcidin, is induced by lipopolysaccharide (LPS) through the IL-6/STAT3 pathway in the cortex and hippocampus. In this paradigm, activated glial cells are the source of IL-6, which was essential in the iron overload-activated apoptosis of neurons. Disrupting astrocyte hepcidin expression prevented the apoptosis of neurons, which were able to maintain levels of FPN1 adequate to avoid iron accumulation. Together, our data are consistent with a model whereby inflammation initiates an intercellular signaling cascade in which activated microglia, through IL-6 signaling, stimulate astrocytes to release hepcidin which, in turn, signals to neurons, via hepcidin, to prevent their iron release. Such a pathway is relevant to NDs in that it links inflammation, microglia and astrocytes to neuronal damage.