Cellular iron depletion enhances behavioral rhythm by limiting brain Per1 expression in mice.

AIMS: Disturbances in the circadian rhythm are positively correlated with the processes of aging and related neurodegenerative diseases, which are also associated with brain iron accumulation. However, the role of brain iron in regulating the biological rhythm is poorly understood. In this study, we investigated the impact of brain iron levels on the spontaneous locomotor activity of mice with altered brain iron levels and further explored the potential mechanisms governing these effects in vitro. RESULTS: Our results revealed that conditional knockout of ferroportin 1 (Fpn1) in cerebral microvascular endothelial cells led to brain iron deficiency, subsequently resulting in enhanced locomotor activity and increased expression of clock genes, including the circadian locomotor output cycles kaput protein (Clock) and brain and muscle ARNT-like 1 (Bmal1). Concomitantly, the levels of period circadian regulator 1 (PER1), which functions as a transcriptional repressor in regulating biological rhythm, were decreased. Conversely, the elevated brain iron levels in APP/PS1 mice inhibited autonomous rhythmic activity. Additionally, our findings demonstrate a significant decrease in serum melatonin levels in Fpn1cdh5 -CKO mice compared with the Fpn1flox/flox group. In contrast, APP/PS1 mice with brain iron deposition exhibited higher serum melatonin levels than the WT group. Furthermore, in the human glioma cell line, U251, we observed reduced PER1 expression upon iron limitation by deferoxamine (DFO; iron chelator) or endogenous overexpression of FPN1. When U251 cells were made iron-replete by supplementation with ferric ammonium citrate (FAC) or increased iron import through transferrin receptor 1 (TfR1) overexpression, PER1 protein levels were increased. Additionally, we obtained similar results to U251 cells in mouse cerebellar astrocytes (MA-c), where we collected cells at different time points to investigate the rhythmic expression of core clock genes and the impact of DFO or FAC treatment on PER1 protein levels. CONCLUSION: These findings collectively suggest that altered iron levels influence the circadian rhythm by regulating PER1 expression and thereby modulating the molecular circadian clock. In conclusion, our study identifies the regulation of brain iron levels as a potential new target for treating age-related disruptions in the circadian rhythm.

Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth.

Transmembrane serine protease 6 (Tmprss6) has been correlated with the occurrence and progression of tumors, but any specific molecular mechanism linking the enzyme to oncogenesis has remained elusive thus far. In the present study, we found that Tmprss6 markedly inhibited mouse neuroblastoma N2a (neuro-2a) cell proliferation and tumor growth in nude mice. Tmprss6 inhibits Smad1/5/8 phosphorylation by cleaving the bone morphogenetic protein (BMP) co-receptor, hemojuvelin (HJV). Ordinarily, phosphorylated Smad1/5/8 binds to Smad4 for nuclear translocation, which stimulates the expression of hepcidin, ultimately decreasing the export of iron through ferroportin 1 (FPN1). The decrease in cellular iron levels in neuro-2a cells with elevated Tmprss6 expression limited the availability of the metal forribo nucleotide reductase activity, thereby arresting the cell cycle prior to S phase. Interestingly, Smad4 promoted nuclear translocation of activating transcription factor 3 (ATF3) to activate the p38 mitogen-activated protein kinases signaling pathway by binding to ATF3, inducing apoptosis of neuro-2a cells and inhibiting tumor growth. Disruption of ATF3 expression significantly decreased apoptosis in Tmprss6 overexpressed neuro-2a cells. Our study describes a mechanism whereby Tmprss6 regulates the cell cycle and apoptosis. Thus, we propose Tmprss6 as a candidate target for inhibiting neuronal tumor growth.

Hepcidin deficiency impairs hippocampal neurogenesis and mediates brain atrophy and memory decline in mice.

BACKGROUND: Hepcidin is the master regulator of iron homeostasis. Hepcidin downregulation has been demonstrated in the brains of Alzheimer's disease (AD) patients. However, the mechanism underlying the role of hepcidin downregulation in cognitive impairment has not been elucidated. METHODS: In the present study, we generated GFAP-Cre-mediated hepcidin conditional knockout mice (HampGFAP cKO) to explore the effect of hepcidin deficiency on hippocampal structure and neurocognition. RESULTS: We found that the HampGFAP cKO mice developed AD-like brain atrophy and memory deficits. In particular, the weight of the hippocampus and the number of granule neurons in the dentate gyrus were significantly reduced. Further investigation demonstrated that the morphological change in the hippocampus of HampGFAP cKO mice was attributed to impaired neurogenesis caused by decreased proliferation of neural stem cells. Regarding the molecular mechanism, increased iron content after depletion of hepcidin followed by an elevated level of the inflammatory factor tumor necrosis factor-α accounted for the impairment of hippocampal neurogenesis in HampGFAP cKO mice. These observations were further verified in GFAP promoter-driven hepcidin knockdown mice and in Nestin-Cre-mediated hepcidin conditional knockout mice. CONCLUSIONS: The present findings demonstrated a critical role for hepcidin in hippocampal neurogenesis and validated the importance of iron and associated inflammatory cytokines as key modulators of neurodevelopment, providing insights into the potential pathogenesis of cognitive dysfunction and related treatments.

Iron overload suppresses hippocampal neurogenesis in adult mice: Implication for iron dysregulation-linked neurological diseases.

AIMS: Adult hippocampal neurogenesis is an important player in brain homeostasis and its impairment participates in neurological diseases. Iron overload has emerged as an irreversible factor of brain aging, and is also closely related to degenerative disorders, including cognitive dysfunction. However, whether brain iron overload alters hippocampal neurogenesis has not been reported. We investigated the effect of elevated iron content on adult hippocampal neurogenesis and explored the underlying mechanism. METHODS: Mouse models with hippocampal iron overload were generated. Neurogenesis in hippocampus and expression levels of related molecules were assessed. RESULTS: Iron accumulation in hippocampus remarkably impaired the differentiation of neural stem cells, resulting in a significant decrease in newborn neurons. The damage was possibly attributed to iron-induced downregulation of proprotein convertase furin and subsequently decreased maturation of brain-derived neurotrophic factor (BDNF), thus contributing to memory decline and anxiety-like behavior of mice. Supportively, knockdown of furin indeed suppressed hippocampal neurogenesis, while furin overexpression restored the impairment. CONCLUSION: These findings demonstrated that iron overload damaged hippocampal neurogenesis likely via iron-furin-BDNF pathway. This study provides new insights into potential mechanisms on iron-induced neurotoxicity and the causes of neurogenesis injury and renders modulating iron homeostasis and furin expression as novel therapeutic strategies for treatment of neurological diseases.

Brain Iron Homeostasis and Mental Disorders.

Iron plays an essential role in various physiological processes. A disruption in iron homeostasis can lead to severe consequences, including impaired neurodevelopment, neurodegenerative disorders, stroke, and cancer. Interestingly, the link between mental health disorders and iron homeostasis has not received significant attention. Therefore, our understanding of iron metabolism in the context of psychological diseases is incomplete. In this review, we aim to discuss the pathologies and potential mechanisms that relate to iron homeostasis in associated mental disorders. We propose the hypothesis that maintaining brain iron homeostasis can support neuronal physiological functions by impacting key enzymatic activities during neurotransmission, redox balance, and myelination. In conclusion, our review highlights the importance of investigating the relationship between trace element nutrition and the pathological process of mental disorders, focusing on iron. This nutritional perspective can offer valuable insights for the clinical treatment of mental disorders.

Iron Metabolism, Redox Balance and Neurological Diseases.

Iron is essential for life, and the dysregulation of iron homeostasis can lead to severe pathological changes in the neurological system [...].

Brain Iron Metabolism, Redox Balance and Neurological Diseases.

The incidence of neurological diseases, such as Parkinson's disease, Alzheimer's disease and stroke, is increasing. An increasing number of studies have correlated these diseases with brain iron overload and the resulting oxidative damage. Brain iron deficiency has also been closely linked to neurodevelopment. These neurological disorders seriously affect the physical and mental health of patients and bring heavy economic burdens to families and society. Therefore, it is important to maintain brain iron homeostasis and to understand the mechanism of brain iron disorders affecting reactive oxygen species (ROS) balance, resulting in neural damage, cell death and, ultimately, leading to the development of disease. Evidence has shown that many therapies targeting brain iron and ROS imbalances have good preventive and therapeutic effects on neurological diseases. This review highlights the molecular mechanisms, pathogenesis and treatment strategies of brain iron metabolism disorders in neurological diseases.

CHIR99021 Maintenance of the Cell Stemness by Regulating Cellular Iron Metabolism.

CHIR99021 is an aminopyrimidine derivative, which can efficiently inhibit the activity of glycogen synthesis kinase 3α (GSK-3α) and GSK-3ß. As an essential component of stem cell culture medium, it plays an important role in maintaining cell stemness. However, the mechanism of its role is not fully understood. In the present study, we first found that removal of CHIR99021 from embryonic stem cell culture medium reduced iron storage in mouse embryonic stem cells (mESCs). CHIR99021-treated Neuro-2a cells led to an upregulation of ferritin expression and an increase in intracellular iron levels, along with GSK3ß inhibition and Wnt/GSK-3ß/ß-catenin pathway activation. In addition, iron treatment activated the classical Wnt pathway by affecting the expression of ß-catenin in the Neuro-2a cells. Our data link the role of iron in the maintenance of cell stemness via the Wnt/GSK-3ß/ß-catenin signaling pathway, and identify intermediate molecules, including Steap1, Bola2, and Kdm6bos, which may mediate the upregulation of ferritin expression by CHIR99021. These findings reveal novel mechanisms of the maintenance of cell stemness and differentiation and provide a theoretical basis for the development of new strategies in stem cell treatment in disease.

Hepcidin is upregulated and is a potential therapeutic target associated with immunity in glioma.

Background: Glioma is the most common primary malignant brain tumor with high mortality and poor prognosis. Hepcidin is a fascinating iron metabolism regulator. However, the prognostic value of hepcidin HAMP in gliomas and its correlation with immune cell infiltration remain unclear. Here, we comprehensively elucidate the prognostic value and potential role of hepcidin in gliomas. Methods: Hepcidin gene expression and clinical characteristics in glioma were analyzed using the CGGA, TCGA, Rembrandt and Gravendeel glioma databases. A survival analysis was conducted using Kaplan-Meier and Cox regression analyses. A gene set enrichment analysis (GSEA) was conducted to select the pathways significantly enriched for hepcidin associations. The correlations between hepcidin and immune cell infiltration and immunotherapy were analyzed using network platforms such as CIBERSORT and TIMER. Results: In glioma tissues, the expression of hepcidin was significantly increased. High hepcidin expression is related to grade, age, PRS type, IDH mutation, chemotherapy status and 1p19q codeletion status, which significantly indicates the poor prognosis of glioma patients. Hepcidin can be used as an independent prognostic factor for glioma through the multivariate COX regression analysis. The results of Gene Ontology (GO), Kyoto Encyclopedia of Gene and Genome (KEGG) and gene set enrichment analysis (GSEA) indicated that hepcidin was involved in the immune response. In addition, hepcidin expression was positively correlated with the degree of immune cell infiltration, the expression of various immune cell markers and the efficacy of immunotherapy. Conclusion: Our results indicate that hepcidin can be used as a candidate biomarker to judge the prognosis and immune cell invasion of gliomas.

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.

The emerging role of furin in neurodegenerative and neuropsychiatric diseases.

Furin is an important mammalian proprotein convertase that catalyzes the proteolytic maturation of a variety of prohormones and proproteins in the secretory pathway. In the brain, the substrates of furin include the proproteins of growth factors, receptors and enzymes. Emerging evidence, such as reduced FURIN mRNA expression in the brains of Alzheimer's disease patients or schizophrenia patients, has implicated a crucial role of furin in the pathophysiology of neurodegenerative and neuropsychiatric diseases. Currently, compared to cancer and infectious diseases, the aberrant expression of furin and its pharmaceutical potentials in neurological diseases remain poorly understood. In this article, we provide an overview on the physiological roles of furin and its substrates in the brain, summarize the deregulation of furin expression and its effects in neurodegenerative and neuropsychiatric disorders, and discuss the implications and current approaches that target furin for therapeutic interventions. This review may expedite future studies to clarify the molecular mechanisms of furin deregulation and involvement in the pathogenesis of neurodegenerative and neuropsychiatric diseases, and to develop new diagnosis and treatment strategies for these diseases.

Caffeine Decreases Hepcidin Expression to Alleviate Aberrant Iron Metabolism under Inflammation by Regulating the IL-6/STAT3 Pathway.

Caffeine is well-known as a psychostimulant, and it can also be beneficial in numerous diseases such as diabetes and different types of cancer. Previous studies have shown that caffeine can have a protective role in bacterial infection-induced inflammation and hyperoxia-mediated pulmonary inflammation. Hepcidin, which is regulated by the IL-6/STAT3 inflammation pathway, is a peptide hormone that maintains systemic iron homeostasis. We hypothesized that caffeine's effects on inflammation may also influence hepcidin production and therefore systemic iron metabolism. To this end, we treated 2-month-old mice with caffeine by daily intragastric administration for 7 days, administering intraperitoneal LPS after the final caffeine treatment. Twelve hours after LPS treatment the mice were euthanized, and tissues were collected. We found that caffeine decreased hepatic hepcidin expression and attenuated LPS-induced hepatic hepcidin overexpression. IL-6 expression and STAT3 phosphorylation were also reduced upon caffeine administration. Additionally, hepatic and splenic FPN1 levels increased after caffeine treatment, leading to lower iron levels in liver and spleen tissues and higher iron levels in serum. Caffeine also prevented the increase in spleen weight and decrease in body weight after LPS treatment. Together, our findings suggest that caffeine decreases hepcidin expression via inhibiting inflammation and the activation of the IL-6/STAT3 pathway, thus presenting an attractive, potential therapeutic for the treatment of anemia of inflammation.

Influence of prazosin on systemic iron levels and the associated iron metabolic alterations in spontaneously hypertensive rats.

The relationship between cardiovascular diseases and iron disorders has gained increasing attention; however, the effects of hypotensive drugs on iron metabolic alterations in hypertension are not well understood. The purpose of this study was to investigate iron metabolic changes after prazosin treatment of spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Our second objective was to examine the effects of hypertension and anti-hypertensive drugs on bone formation and resorption. SHRs and WKY rats were randomized into either prazosin-treated groups (WKY + PZ and SHR + PZ) or untreated groups (WKY and SHR). After 7 days of intragastric prazosin administration, the rats were sacrificed for analysis; blood samples and organs (the duodenum, liver, kidneys, spleen, and femur) were collected. Both WKY + PZ and SHR groups exhibited iron deficiency in the serum and liver. Prazosin increased the iron levels in the bone tissue of SHRs. Prazosin stimulated the expression of hepcidin mRNA in the liver of SHRs and inhibited the expression of this iron-regulatory hormone in WKY rats. FPN1 expression in the duodenum was increased significantly in SHRs, however markedly decreased after prazosin treatment. The expression of TLR4 and Ctsk was enhanced in the bone tissue of SHRs, whereas CLC-7 expression was inhibited. Both hypotension and hypertension can lead to iron deficiency. Treatment with prazosin restored iron homeostasis in SHRs. The inverse impacts of prazosin on hepatic hepcidin expression in SHRs versus WKY rats indicates differing iron regulatory mechanisms between hypertensive and normal animals. The osteoclast activity was found to be enhanced in SHRs. Further study is needed to address whether the changes in osteoblast and osteoclast activity in SHRs correlates with the effects on iron metabolism.

Overexpression of Mitochondrial Ferritin Enhances Blood-Brain Barrier Integrity Following Ischemic Stroke in Mice by Maintaining Iron Homeostasis in Endothelial Cells.

Blood-brain barrier (BBB) breakdown, a characteristic feature of ischemic stroke, contributes to poor patient outcomes. Brain microvascular endothelial cells (BMVECs) are a key component of the BBB and dysfunction or death of these cells following cerebral ischemia reperfusion (I/R) injury can disrupt the BBB, leading to leukocyte infiltration, brain edema and intracerebral hemorrhage. We previously demonstrated that mitochondrial ferritin (FtMt) can alleviate I/R-induced neuronal ferroptosis by inhibiting inflammation-regulated iron deposition. However, whether FtMt is involved in BBB disruption during cerebral I/R is still unknown. In the present study, we found that FtMt expression in BMVECs is upregulated after I/R and overexpression of FtMt attenuates I/R-induced BBB disruption. Mechanistically, we found that FtMt prevents tight junction loss and apoptosis by inhibiting iron dysregulation and reactive oxygen species (ROS) accumulation in I/R-treated BMVECs. Chelating excess iron with deferoxamine alleviates apoptosis in the brain endothelial cell line bEnd.3 under oxygen glucose deprivation followed by reoxygenation (OGD/R) insult. In summary, our data identify a previously unexplored effect for FtMt in the BBB and provide evidence that iron-mediated oxidative stress in BMVECs is an early cause of BMVECs damage and BBB breakdown in ischemic stroke.

Hippocampal Iron Accumulation Impairs Synapses and Memory via Suppressing Furin Expression and Downregulating BDNF Maturation.

Brain iron overload is positively correlated with the pathogenesis of Alzheimer's disease (AD). However, the role of iron in AD pathology is not completely understood. Furin is the first identified mammalian proprotein convertase that catalyzes the proteolytic maturation of large numbers of prohormones and proproteins. The correlation between altered furin expression and AD pathology has been suggested, but the underlying mechanism remains to be clarified. Here, we found that the expression of furin in the hippocampus of Alzheimer's model APP/PS1 mice was significantly reduced, and we demonstrated that the reduction of furin was directly caused by hippocampal iron overload using wild-type mice with intrahippocampal injection of iron. In cultured neuronal cells, this suppression effect was observed as transcriptional inhibition. Regarding the changes of furin-mediated activities caused by hippocampal iron overload, we found that the maturation of brain-derived neurotrophic factor (BDNF) was impeded and the expression levels of synaptogenesis-related proteins were downregulated, leading to cognitive decline. Furthermore, iron chelation or furin overexpression in the hippocampus of APP/PS1 mice increased furin expression, restored synapse plasticity, and ameliorated cognitive decline. Therefore, the inhibitory effect of hippocampal iron accumulation on furin transcription may be an important pathway involved in iron-mediated synapse damage and memory loss in AD. This study provides new insights into the molecular mechanisms of the toxic effects of iron in neurons and AD pathophysiology and renders furin as a potential target for treatment of iron overload-related neurodegenerative diseases.

Osteoblast Derived Exosomes Alleviate Radiation- Induced Hematopoietic Injury.

As hematopoietic stem cells can differentiate into all hematopoietic lineages, mitigating the damage to hematopoietic stem cells is important for recovery from overdose radiation injury. Cells in bone marrow microenvironment are essential for hematopoietic stem cells maintenance and protection, and many of the paracrine mediators have been discovered in shaping hematopoietic function. Several recent reports support exosomes as effective regulators of hematopoietic stem cells, but the role of osteoblast derived exosomes in hematopoietic stem cells protection is less understood. Here, we investigated that osteoblast derived exosomes could alleviate radiation damage to hematopoietic stem cells. We show that intravenous injection of osteoblast derived exosomes promoted WBC, lymphocyte, monocyte and hematopoietic stem cells recovery after irradiation significantly. By sequencing osteoblast derived exosomes derived miRNAs and verified in vitro, we identified miR-21 is involved in hematopoietic stem cells protection via targeting PDCD4. Collectively, our data demonstrate that osteoblast derived exosomes derived miR-21 is a resultful regulator to radio-protection of hematopoietic stem cells and provide a new strategy for reducing radiation induced hematopoietic injury.

Iron promotes neurological function recovery in mice with ischemic stroke through endogenous repair mechanisms.

The endogenous repair mechanisms play an important role in the recovery of nerve function after stroke, such as gliosis, synaptic plasticity, remyelination and nerve regeneration. Iron is the most abundant trace metal element in the brain and plays a crucial role in the maintenance of normal cerebral function. It is an important coenzyme factor in the process of cell metabolism, DNA synthesis, purine catabolism and neurotransmitter synthesis and decomposition. However, it is unclear what role iron plays in the long-term recovery of neurological function after stroke. In this study, we first observed that changes in iron metabolism occurred during neurological function recovery in the mice with distal middle cerebral artery occlusion (dMCAO). Our data showed that plasticity changes due to endogenous repair mechanisms resulted in improvements in cerebral cortex function. These changes involved gliosis, synaptic function reconstruction, remyelination, and activation of neural stem cells. In order to examine the potential role of iron, we synthesized liposomal-encapsulated deferoxamine (DFO) nanoparticles to further explore the effect and the mechanism of iron on the recovery of neurological function in dMCAO mice. Our results showed that liposome-DFO decreased iron deposition and reversed plasticity changes in cerebral cortex function after stroke, which delayed neurological function recovery. This experiment shows that the increasing iron level promotes endogenous repair in ischemic stroke. Our finding reveals the change regularity of iron and emphasizes the beneficial role of iron in the recovery process of neurological function, which provides an important basis for the prevention and/or treatment of ischemia-reperfusion and recovery after stroke.

3' untranslated region of Ckip-1 inhibits cardiac hypertrophy independently of its cognate protein.

AIMS: 3' untranslated region (3' UTR) of mRNA is more conserved than other non-coding sequences in vertebrate genomes, and its sequence space has substantially expanded during the evolution of higher organisms, which substantiates their significance in biological regulation. However, the independent role of 3' UTR in cardiovascular disease was largely unknown. METHODS AND RESULTS: Using bioinformatics, RNA fluorescent in situ hybridization and quantitative real-time polymerase chain reaction, we found that 3' UTR and coding sequence regions of Ckip-1 mRNA exhibited diverse expression and localization in cardiomyocytes. We generated cardiac-specific Ckip-1 3' UTR overexpression mice under wild type and casein kinase 2 interacting protein-1 (CKIP-1) knockout background. Cardiac remodelling was assessed by histological, echocardiography, and molecular analyses at 4 weeks after transverse aortic constriction (TAC) surgery. The results showed that cardiac Ckip-1 3' UTR significantly inhibited TAC-induced cardiac hypertrophy independent of CKIP-1 protein. To determine the mechanism of Ckip-1 3' UTR in cardiac hypertrophy, we performed transcriptome and metabolomics analyses, RNA immunoprecipitation, biotin-based RNA pull-down, and reporter gene assays. We found that Ckip-1 3' UTR promoted fatty acid metabolism through AMPK-PPARα-CPT1b axis, leading to its protection against pathological cardiac hypertrophy. Moreover, Ckip-1 3' UTR RNA therapy using adeno-associated virus obviously alleviates cardiac hypertrophy and improves heart function. CONCLUSIONS: These findings disclose that Ckip-1 3' UTR inhibits cardiac hypertrophy independently of its cognate protein. Ckip-1 3' UTR is an effective RNA-based therapy tool for treating cardiac hypertrophy and heart failure.

Calcitonin increases hepatic hepcidin expression through the BMP6 of kidney in mice.

BACKGROUND: Osteoporosis is frequently accompanied by iron disorders. Calcitonin (CT) was approved as a clinical drug to treat osteoporosis. Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin deficiency leads to iron overload diseases. This study was aimed at investigating the effect of CT on hepatic hepcidin and the mechanism by which CT modulates hepatic hepcidin pathways and iron metabolism. METHOD: RT-PCR, Western blot, ELISA and siRNA were used to detect the effect of CT on iron metabolism in vivo and in vitro. In addition, the regulatory signal molecules of hepcidin were measured to explore the molecular mechanism of its regulation. RESULTS: The results showed that CT strongly increased hepcidin expression and altered iron homeostasis, after mice were intraperitoneal injection of CT. In response to CT administration, BMP6 level in kidney and the serum BMP6 was increased significantly. The phosphorylation of Smad1/5/8 proteins in liver was increased at 3 h and 6 h. Moreover, the Bmp inhibitor LDN-193,189 pretreatment significantly attenuated the CT-mediated increases in phosphorylated Smad1/5/8 and Hamp1 mRNA levels. Calcitonin receptor (CTR) siRNA transfection significant suppressed the role of CT on BMP6 expression in Caki-1 cells. CONCLUSION: Our results suggest that CT strongly induces hepcidin expression and affected iron metabolism. It will provide a new strategy for the treatment of calcium iron related diseases.

Targeting E3 Ubiquitin Ligase WWP1 Prevents Cardiac Hypertrophy Through Destabilizing DVL2 via Inhibition of K27-Linked Ubiquitination.

BACKGROUND: Without adequate treatment, pathological cardiac hypertrophy induced by sustained pressure overload eventually leads to heart failure. WWP1 (WW domain-containing E3 ubiquitin protein ligase 1) is an important regulator of aging-related pathologies, including cancer and cardiovascular diseases. However, the role of WWP1 in pressure overload-induced cardiac remodeling and heart failure is yet to be determined. METHODS: To examine the correlation of WWP1 with hypertrophy, we analyzed WWP1 expression in patients with heart failure and mice subjected to transverse aortic constriction (TAC) by Western blotting and immunohistochemical staining. TAC surgery was performed on WWP1 knockout mice to assess the role of WWP1 in cardiac hypertrophy, heart function was examined by echocardiography, and related cellular and molecular markers were examined. Mass spectrometry and coimmunoprecipitation assays were conducted to identify the proteins that interacted with WWP1. Pulse-chase assay, ubiquitination assay, reporter gene assay, and an in vivo mouse model via AAV9 (adeno-associated virus serotype 9) were used to explore the mechanisms by which WWP1 regulates cardiac remodeling. AAV9 carrying cardiac troponin T (cTnT) promoter-driven small hairpin RNA targeting WWP1 (AAV9-cTnT-shWWP1) was administered to investigate its rescue role in TAC-induced cardiac dysfunction. RESULTS: The WWP1 level was significantly increased in the hypertrophic hearts from patients with heart failure and mice subjected to TAC. The results of echocardiography and histology demonstrated that WWP1 knockout protected the heart from TAC-induced hypertrophy. There was a direct interaction between WWP1 and DVL2 (disheveled segment polarity protein 2). DVL2 was stabilized by WWP1-mediated K27-linked polyubiquitination. The role of WWP1 in pressure overload-induced cardiac hypertrophy was mediated by the DVL2/CaMKII/HDAC4/MEF2C signaling pathway. Therapeutic targeting WWP1 almost abolished TAC induced heart dysfunction, suggesting WWP1 as a potential target for treating cardiac hypertrophy and failure. CONCLUSIONS: We identified WWP1 as a key therapeutic target for pressure overload induced cardiac remodeling. We also found a novel mechanism regulated by WWP1. WWP1 promotes atypical K27-linked ubiquitin multichain assembly on DVL2 and exacerbates cardiac hypertrophy by the DVL2/CaMKII/HDAC4/MEF2C pathway.