Hephaestin Research Articles

TCONS_00025035-miR-101-UROS is potentially involved in the regulation of heme synthesis pathway and influences mantle melanin deposition by targeting porphyrin in Manila clam (Ruditapes philippinarum)

Shell color is an important economic trait and one of the target traits in breeding and production. Non-coding RNA (ncRNA) refers to RNA molecules transcribed from the genome and do not encoding proteins, which can regulate the expression of target genes after transcription and participate in the regulation of many important traits, such as the formation of shell color and body color. In this study, we detected the porphyrins in the shells of three Manila clams with different shell colors, explored the expression pattern and function of Uroporphyrinogen III synthetase (UROS) in the shell color pigmentation of Ruditapes philippinarum, and found that it might be involved in the synthesis of porphyrins and potentially in the synthesis of melanin. The results showed that the expression levels of heme synthesis-related genes such as UROS, Uroporphyrinogen decarboxylase (UROD), Ferrochelatase (FECH), Hephaestin (HEPH), and pigment synthesis-related genes (Peroxidasin PXDN) in the positive group were significantly reduced compared with the control group after injection of UROS dsRNA, indicating that UROS plays a crucial role in the porphyrin synthesis pathway. Additionally, transmission electron microscopy and melanin extraction experiments also proved that it might participate in the synthesis of melanin. We further explored and verified the relationship between TCONS_00025035-miR-101-UROS and identified the changes in the expression level of UROS through RNA interference and injection of miR-101 antagomir, respectively. Our results imply that miR-101 antagonists affect the expression of UROS. Furthermore, dual-luciferase reporter gene experiments confirmed the relationship between TCON_00025035, miR-101, and UROS. The regulatory relationship between TCONS_00025035 and miR-101 is negative, and the regulatory relationship between miR-101 and UROS is also negative. In summary, we verified the function of UROS through RNA interference, qPCR, in situ hybridization, and melanin content detection. We speculated that there was a negative relationship between miR-101 and UROS, and there was also a negative relationship between TCONS_00025035 and miR-101. TCONS_00025035 might regulate UROS through the regulation of miR-101, and UROS might also regulate other pigmentation-related genes and affect the formation of pigments, thereby influencing porphyrin and melanin formation in Manila clam.

Whole-genome resequencing identifies candidate genes and SNPs in genomic regions associated with shell color selection in the Pacific oyster, Crassostrea gigas

The various shell colors of bivalves not only result in a better visual perception but also show great commercial value as a highly potential breeding trait. Four shell color strains of Pacific oyster (Crassostrea gigas) characterized by black (SB), white (SW), gold (SG), and orange (SO) were bred purposefully for more than ten generations. In this study, whole-genome resequencing was performed on 20 oysters from each of the four strains, and a total of 14.67 million single nucleotide polymorphisms (SNPs) were obtained after stringent filtering, with an average sequencing depth of >10×. The population structure analysis provided support for the subdivision of all samples into four major genetic clusters corresponding to shell color phenotypes, indicating a high level of genomic differentiation driven by artificial selection. Then, a composite measure of fixation index (FST) and cross-population composite likelihood ratio (XP-CLR) allowed us to identify 336 to 414 candidate divergent regions (CDRs) in pairwise comparisons of different shell colors, with a total of 480 to 614 corresponding genes distributed. Of particular interest were tyrosinase (TYR) and perilipin2 (PLIN2) occurring with extremely high frequency, which participates in shell color determination by regulating melanin production and carotenoid metabolism, respectively. In addition, two genes, namely porphobilinogen deaminase (PBGD) and hephaestin (HEPH), may have a potential function in the accumulation of specific porphyrin in orange shells, while the kynurenine 3-monooxygenase (KMO) gene, known to be involved in ommochrome biosynthesis, may contribute to the darker coloration in black shells. Further, a total of 304 SNPs were extracted from the exons of candidate genes mentioned above, of which 25 loci had highly significant differences among populations (p-value ≤0.01). After Sanger sequencing in another population, 14 SNPs were successfully validated as specific loci closely linked to different shell colors of black, white, gold, and orange, respectively. All in all, the results of this study shed light on the molecular basis of pigmentation and facilitate the selective breeding of C. gigas with desired phenotypic color.

Abstract A014: Role of stromal CD10 expression in progression of endometrial and endometriosis-associated cancers

Abstract Endometriosis-associated carcinomas (EACs) and endometrial malignancies have many commonalities yet their pathophysiology, particularly those with high-risk histology such as clear cell, is understudied. EACs are histologically and clinically distinct from other gynecological cancers due to their unique origin within endometriosis. The endometriotic microenvironment is uniquely hypoxic with high levels of iron due to recurrent menstrual cycling, and this iron-rich microenvironment has been linked to promoting carcinogenesis through iron-induced oxidative stress. However, the mechanism by which the endometriotic microenvironment contributes to EAC tumorigenesis and how this compares with endometrial cancer is unknown. Most of our current knowledge of EACs is built on either epidemiologic analyses or correlative studies. Additionally, most studies of the endometriotic microenvironment primarily focus on the epithelial component while the role of the stroma has been largely unexplored. Mesenchymal stem cells (MSC) are a critical component of the stromal microenvironment. Recently, we identified a key role of endometriosis derived MSCs (enMSCs) in altering ovarian clear cell carcinoma iron regulation. Interestingly, enMSCs resemble the primary endometrium derived MSCs, which suggested their role in endometrial cancer. This stromal subset is characterized by the loss of surface CD10 expression. It is known that cancer cells, particularly clear cell carcinoma (CCC), have a unique requirement for high levels of iron. We hypothesize that CD10 negative enMSCs promote growth of EAC and endometrial cancer through iron regulation. MSCs were isolated from primary human benign endometriosis, benign endometrium, or endometrial cancer. Flow cytometry was used to measure surface CD10 expression. We investigated the role of CD10 negative enMSCs on CCC iron regulation. Our results showed that CD10 negative enMSCs promote CCC growth by exporting iron through the upregulation of Hephaestin (HEPH) and Ferroportin (FPO) which then led to increase labile iron pool (LIP) in CCC cells. However, increasing the LIP does not occur with iron supplementation alone, this indicates the critical role of CD10 negative enMSCs in regulating iron handling in CCC cells. Additionally, our data showed that CD10 negative enMSCs alter the balance of Ferritin L and Ferritin H- the main iron storage proteins- in CCC cells. Mechanistically, our preliminary data showed that CD10 negative enMSCs directly donate Ferritin L into CCC cells. Further, CD10 negative enMSCs enhance Ferritin H degradation through the upregulation of ferritinophagy in CCC cells. In conclusion, our results indicate there is a sub-population of enMSCs, marked by the loss of CD10 expression, that enhances EAC and endometrial cancer cell growth through iron regulation. This highlights the existence of a tumor-promoting stromal cell within both the endometrium and endometriosis which can be capitalized on as an “Achilles' heel” to uncover novel treatment targets for EAC and endometrial cancer. Citation Format: Huda I. Atiya, Lan G. Coffman. Role of stromal CD10 expression in progression of endometrial and endometriosis-associated cancers [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A014.

Different iron sources affected haemoglobin and myoglobin synthesis, and gene expression related to iron metabolism in skeletal muscle in piglets

ABSTRACT This study was carried out to investigate the effect of different iron sources on haemoglobin and myoglobin (Mb) synthesis, and the mRNA expression of the key genes related to iron metabolism in the skeletal muscle of piglets. Forty-eight piglets were assigned into four treatments including control group, ferrous sulphate (FeSO4) group, ferrous glycinate (Fe-Gly) group and amino acid-Fe(II)-chelator complexes group. The results showed that iron supplementation increased Mb concentration in muscles (P < 0.05). In addition, iron supplementation increased hephaestin (HEPH) expression in the longissimus dorsi muscle (P < 0.1), decreased transferrin receptor 1 (TFR1) expression in the longissimus dorsi muscle (P < 0.05) and increased lipocalin 2 (Lcn2) expression in gastrocnemius muscle, respectively (P < 0.05). In summary, compared with high-dose inorganic iron, low-dose organic iron supplements have the same effects in haemoglobin and myoglobin biosynthesis, and mobilize iron in muscles into circulation.

Quercetin Inhibits Hephaestin Expression and Iron Transport in Intestinal Cells: Possible Role of PI3K Pathway

Previous studies demonstrated that quercetin, a polyphenolic compound, inhibits the transport of iron by down-regulation of ferroportin (FPN1), an iron export protein. We have previously demonstrated that activation of the PI3K signaling pathway by zinc stimulates the intestinal iron uptake and transport by stimulating the expression of iron regulatory protein 2 (IRP2) dependent divalent metal iron transporter 1 (DMT1, apical iron transporter) expression and caudal-related homeobox transcription factor 2 (CDX2) dependent hephaestin (HEPH, basolateral ferroxidase required for iron oxidation) expression, respectively. Since polyphenols are antagonists of the PI3K pathway, we hypothesized that quercetin might inhibit basolateral iron transport via the down-regulation of hephaestin (HEPH). Here in we investigated the effect of quercetin on iron uptake, transport, and expression of iron transporters in intestinal cells. In differentiated Caco-2 cells grown on permeable supports, quercetin inhibited the basolateral iron transport while increasing the iron uptake, possibly due to higher cellular retention. Further, quercetin down-regulated the protein and mRNA expression of HEPH and FPN1 but not that of IRP2 or DMT1. In addition, quercetin also abrogated the zinc-induced Akt, CDX2 phosphorylation, and HEPH expression. Together these results suggest that inhibition of iron transport by quercetin is mediated via the down-regulation of CDX2-dependent HEPH expression via inhibition of the PI3K pathway.

Zinc induces hephaestin expression via a PI3K-CDX2 dependent mechanism to regulate iron transport in intestinal Caco-2 cells

Zinc stimulates intestinal iron absorption via induction of divalent metal ion transporter (DMT1) and hephaestin (HEPH). While the increase in DMT1 is mediated via a PI3K/IPR2 axis, the mechanisms of Zn-induced HEPH expression downstream of PI3K remain elusive. In the current study we probed the role of Caudal-related homeobox transcription factor-2 (CDX2) on Zn-induced HEPH expression. Zn treatment of Caco-2 cells increased CDX2 phosphorylation and HEPH protein and mRNA expression. siRNA-silencing of CDX2 inhibited Zn-induced HEPH expression. LY294002, an antagonist of PI3K inhibited Zn-induced phosphorylation of CDX2, and downstream HEPH expression. These results suggest that increased expression of HEPH in intestinal cells following Zn treatment is mediated via a PI3K-CDX2 pathway.

IRE-dependent Regulation of Intestinal Dmt1 Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress.

IRE-dependent Regulation of Intestinal Dmt1 Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress.

The biology of mammalian multi-copper ferroxidases.

The mammalian multicopper ferroxidases (MCFs) ceruloplasmin (CP), hephaestin (HEPH) and zyklopen (ZP) comprise a family of conserved enzymes that are essential for body iron homeostasis. Each of these enzymes contains six biosynthetically incorporated copper atoms which act as intermediate electron acceptors, and the oxidation of iron is associated with the four electron reduction of dioxygen to generate two water molecules. CP occurs in both a secreted and GPI-linked (membrane-bound) form, while HEPH and ZP each contain a single C-terminal transmembrane domain. These enzymes function to ensure the efficient oxidation of iron so that it can be effectively released from tissues via the iron export protein ferroportin and subsequently bound to the iron carrier protein transferrin in the blood. CP is particularly important in facilitating iron release from the liver and central nervous system, HEPH is the major MCF in the small intestine and is critical for dietary iron absorption, and ZP is important for normal hair development. CP and HEPH (and possibly ZP) function in multiple tissues. These proteins also play other (non-iron-related) physiological roles, but many of these are ill-defined. In addition to disrupting iron homeostasis, MCF dysfunction perturbs neurological and immune function, alters cancer susceptibility, and causes hair loss, but, despite their importance, how MCFs co-ordinately maintain body iron homeostasis and perform other functions remains incompletely understood.

The Ferroxidase Hephaestin in Lung Cancer: Pathological Significance and Prognostic Value.

Hephaestin (HEPH) belongs to a group of exocytoplasmic ferroxidases which contribute to cellular iron homeostasis by favouring its export. Down-regulation of HEPH expression, possibly by stimulating cell proliferation due to an increase in iron availability, has shown to correlate with poor survival in breast cancer. The lung is particularly sensitive to iron-induced oxidative stress, given the high oxygen tension present, however, HEPH distribution in lung cancer and its influence on prognosis have not been investigated yet. In this study we explored the prognostic value of HEPH and its expression pattern in the most prevalent histotypes of lung cancers, namely lung adenocarcinoma and lung squamous cell carcinoma. In silico analyses, based on UALCAN, Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier plotter bioinformatics, revealed a significant correlation between higher levels of HEPH expression and favorable prognosis, in both cancer histotypes. Moreover, TIMER web platform showed a statistically significant association between HEPH expression and cell elements belonging to the tumor microenvironment identified as endothelial cells and a subpopulation of cancer-associated fibroblasts, further confirmed by double immunohistochemical labeling with cell type specific markers. Taken together, these data shed a light on the complex mechanisms of local iron handling lung cancer can exploit to support tumorigenesis.

Effect of atrazine on accumulation of iron via the iron transport proteins in the midbrain of SD rats.

Atrazine (ATR), a widely used herbicide that belongs to the triazine class, has detrimental effects on several organ systems. It has also been shown that ATR exposure results in dopaminergic neurotoxicity. However, the mechanism of herbicides causing ferroptosis in neurons is less concerned. So, the present study aimed to investigate the effects of long-term oral exposure to ATR on ferroptosis in adult male rats. In this study, we show that there was a dose-dependent increase in the concentration of iron in the midbrain. Simultaneously, the expression of tyrosine hydroxylase (TH) and Synuclein (α-syn) were altered by the ATR. We carried out miRNA profiling brain tissue in order to identify factors that mediate ferroptosis. We also found that the mRNA and protein expression of the transferrin receptor (TFR), divalent metal transporter 1 (DMT1), hephaestin (HEPH), and ferroportin 1 (Fpn1) in the midbrain were affected by ATR. Based on the current results and previously published data, it is clear that exposure of adult male rats to high doses of ATR leads to iron loading in the midbrain. The long-term adverse effects of ATR on the midbrain have a special relevance after exposure.

MiR-194-5p inhibits SLC40A1 expression to induce cisplatin resistance in ovarian cancer

BackgroundmiR-194-5p has been associated with drug resistance in many cancers. However, the role of miR-194-5p in cisplatin resistance in ovarian cancer is still unclear. Materials and methodsTo study the role and mechanism of miR-194-5p in cisplatin resistance, qRT-PCR was performed to determine the expression of miR-194-5p and SLC40A1 in ovarian cancer. Cell Counting Kit-8 (CCK8) assay was used to analyse cell viability after cisplatin treatment. Dual-luciferase reporter gene assay was performed to examine the relationship between miR-194-5p and SLC40A1. The genes downstream of SLC40A1 were investigated through bioinformatics analysis. ResultsCompared to cisplatin-sensitive ovarian cancer cells, higher miR-194-5p expression and lower SLC40A1 expression were found in cisplatin-resistant ovarian cancer cells. Moreover, this study demonstrated that over-expression of miR-194-5p inhibited SLC40A1 expression, and knockdown of miR-194-5p promoted SLC40A1 expression. In addition, dual-luciferase reporter gene assay further confirmed the negative correlation between miR-194-5p and SLC40A1. Furthermore, we found that over-expression of miR-194-5p resulted in cisplatin resistance. When miR-194-5p and SLC40A1 were simultaneously up-regulated, cisplatin sensitivity increased, while down-regulation of miR-194-5p sensitised ovarian cancer cells to cisplatin. However, when miR-194-5p and SLC40A1 were simultaneously down-regulated, cisplatin sensitivity was decreased. These data suggested that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance. In addition, bioinformatics analysis indicated a positive correlation of SLC40A1 with hephaestin (HEPH), and homeostatic iron regulator (HFE). However, we found that HEPH and HFE were associated with cisplatin resistance, suggesting that their role in drug resistance is induced by miR-194-5p/SLC40A1. ConclusionIn conclusion, we found that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance in ovarian cancer. This study suggests that miR-194-5p could be a potential therapeutic target and a prognostic biomarker for ovarian cancer, with important implications for future research.

Active compounds of herbs ameliorate impaired cognition in APP/PS1 mouse model of Alzheimer's disease.

Alzheimer’s disease (AD), the most common cause of dementia, is a neurodegenerative disorder characterized by amyloid plaque accumulations, intracellular tangles and neuronal loss in certain brain regions. It has been shown that a disturbance of normal iron metabolism contributes to the pathophysiology of AD. However, the mechanism underlying abnormal iron load in the brain of AD patients is unclear. The frontal cortex, an important brain structure for executive function, is one of the regions affected by AD. We investigated the beneficial effects of active compounds of Epimedium, Astragaoside and Puerarin on iron metabolism in the frontal cortex of six-month-old APPswe/PS1ΔE9 (APP/PS1) double transgenic mouse, a model of AD. Treatment with the active compounds reduced cognitive and memory deficits and damaged cell ultrastructure in APP/PS1 mice. These beneficial effects were associated with changes in expression levels of iron metabolism proteins in the frontal cortex, including divalent metal transporter with iron response element (DMT1-with IRE), divalent metal transporter without iron response element (DMT1-without IRE), transferrin (TF) and transferring receptor 1 (TfR1); three release proteins including the exporter ferroportin 1 (Fpn1), ceruloplasmin (CP) and hephaestin (HEPH), one increased storage iron protein ferritin and one iron regulating hormone hepcidin. These findings suggest that the active compounds improve cognition and memory in brain neurodegenerative disorders and these beneficial effects are associated with reduced impairment of iron metabolism. This study may provide a new strategy for developing novel drugs to treat AD.

Quantum Blue Reduces the Severity of Woody Breast Myopathy via Modulation of Oxygen Homeostasis-Related Genes in Broiler Chickens.

The incidence of woody breast (WB) is increasing on a global scale representing a significant welfare problem and economic burden to the poultry industry and for which there is no effective treatment due to its unknown etiology. In this study, using diffuse reflectance spectroscopy (DRS) coupled with iSTAT portable clinical analyzer, we provide evidence that the circulatory- and breast muscle-oxygen homeostasis is dysregulated [low oxygen and hemoglobin (HB) levels] in chickens with WB myopathy compared to healthy counterparts. Molecular analysis showed that blood HB subunit Mu (HBM), Zeta (HBZ), and hephaestin (HEPH) expression were significantly down regulated; however, the expression of the subunit rho of HB beta (HBBR) was upregulated in chicken with WB compared to healthy counterparts. The breast muscle HBBR, HBE, HBZ, and hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) mRNA abundances were significantly down regulated in WB-affected compared to normal birds. The expression of HIF-1α at mRNA and protein levels was significantly induced in breasts of WB-affected compared to unaffected birds confirming a local hypoxic status. The phosphorylated levels of the upstream mediators AKT at Ser473 site, mTOR at Ser2481 site, and PI3K P85 at Tyr458 site, as well as their mRNA levels were significantly increased in breasts of WB-affected birds. In attempt to identify a nutritional strategy to reduce WB incidence, male broiler chicks (Cobb 500, n = 576) were randomly distributed into 48 floor pens and subjected to six treatments (12 birds/pen; 8 pens/treatment): a nutrient adequate control group (PC), the PC supplemented with 0.3% myo-inositol (PC + MI), a negative control (NC) deficient in available P and Ca by 0.15 and 0.16%, respectively, the NC fed with quantum blue (QB) at 500 (NC + 500 FTU), 1,000 (NC + 1,000 FTU), or 2,000 FTU/kg of feed (NC + 2,000 FTU). Although QB-enriched diets did not affect growth performances (FCR and FE), it did reduce the severity of WB by 5% compared to the PC diet. This effect is mediated by reversing the expression profile of oxygen homeostasis-related genes; i.e., significant down regulation of HBBR and upregulation of HBM, HBZ, and HEPH in blood, as well as a significant upregulation of HBA1, HBBR, HBE, HBZ, and PHD2 in breast muscle compared to the positive control.

Mechanisms, mishaps and manipulation of iron uptake

Mechanisms, mishaps and manipulation of iron uptake

Biallelic HEPHL1 variants impair ferroxidase activity and cause an abnormal hair phenotype.

Maintenance of the correct redox status of iron is functionally important for critical biological processes. Multicopper ferroxidases play an important role in oxidizing ferrous iron, released from the cells, into ferric iron, which is subsequently distributed by transferrin. Two well-characterized ferroxidases, ceruloplasmin (CP) and hephaestin (HEPH) facilitate this reaction in different tissues. Recently, a novel ferroxidase, Hephaestin like 1 (HEPHL1), also known as zyklopen, was identified. Here we report a child with compound heterozygous mutations in HEPHL1 (NM_001098672) who presented with abnormal hair (pili torti and trichorrhexis nodosa) and cognitive dysfunction. The maternal missense mutation affected mRNA splicing, leading to skipping of exon 5 and causing an in-frame deletion of 85 amino acids (c.809_1063del; p.Leu271_ala355del). The paternal mutation (c.3176T>C; p.Met1059Thr) changed a highly conserved methionine that is part of a typical type I copper binding site in HEPHL1. We demonstrated that HEPHL1 has ferroxidase activity and that the patient’s two mutations exhibited loss of this ferroxidase activity. Consistent with these findings, the patient’s fibroblasts accumulated intracellular iron and exhibited reduced activity of the copper-dependent enzyme, lysyl oxidase. These results suggest that the patient’s biallelic variants are loss-of-function mutations. Hence, we generated a Hephl1 knockout mouse model that was viable and had curly whiskers, consistent with the hair phenotype in our patient. These results enhance our understanding of the function of HEPHL1 and implicate altered ferroxidase activity in hair growth and hair disorders.

Organ‑specific expression of the divalent ion channel proteins NCKX3, TRPV2, CTR1, ATP7A, IREG1 and HEPH in various canine organs.

Transmembrane cation channels include those for calcium, copper and iron ion transport. Each channel has physiological significance, and all have been associated with disease. However, the comparative study of transcriptional‑translational levels in canine organs has not been previously reported. In the present study, organ‑specific expression of calcium channels, including sodium/potassium/calcium exchanger 3 (NCKX3) and transient receptor potential cation channel subfamilyV member 2 (TRPV2), copper channels, including high affinity copper uptake protein 1 (CTR1) and copper‑transporting ATPase 1 (ATP7A), and iron channels, including iron‑regulated transporter 1 (IREG1) and hephaestin (HEPH) proteins and their mRNAs were examined in the canine duodenum, kidney, spleen and liver. NCKX3 protein expression was highest in the kidney, moderate in the duodenum, and lowest in the spleen and liver, whereas TRPV2 protein was highly expressed in the kidney, duodenum and liver, and was low in the spleen. The CTR1 protein expression level was highest in the liver, followed (in descending order) by the duodenum, kidney and spleen. The ATP7A protein expression level was highest in the duodenum and lowest in the spleen. The IREG1 protein expression level was highest in the liver, followed (in descending order) by the kidney, duodenum and spleen. The HEPH protein level was high in liver, moderate in the duodenum and kidney, and low in the spleen. The results of the immunohistochemistry analysis demonstrated ion channel protein localizations. These results suggested that cation channel proteins are differentially expressed among canine organs, and they may be involved in organ‑specific functions associated with the maintenance of physiological homeostasis.

Severe Iron Metabolism Defects in Mice With Double Knockout of the Multicopper Ferroxidases Hephaestin and Ceruloplasmin.

Background & AimsMulticopper ferroxidases (MCFs) facilitate intestinal iron absorption and systemic iron recycling, likely by a mechanism involving the oxidization of Fe2+ from the iron exporter ferroportin 1 for delivery to the circulating Fe3+ carrier transferrin. Hephaestin (HEPH), the only MCF known to be expressed in enterocytes, aids in the basolateral transfer of dietary iron to the blood. Mice lacking HEPH in the whole body (Heph-/-) or intestine alone (Hephint/int) exhibit defects in dietary iron absorption but still survive and grow. Circulating ceruloplasmin (CP) is the only other known MCF likely to interact with enterocytes. Our aim was to assess the effects of combined deletion of HEPH and CP on intestinal iron absorption and homeostasis in mice.MethodsMice lacking both HEPH and CP (Heph-/-Cp-/-) and mice with whole-body knockout of CP and intestine-specific deletion of HEPH (Hephint/intCp-/-) were generated and phenotyped.ResultsHeph-/-Cp-/- mice were severely anemic and had low serum iron, but they exhibited marked iron loading in duodenal enterocytes, the liver, heart, pancreas, and other tissues. Hephint/intCp-/- mice were moderately anemic (similar to Cp-/- mice) but were iron loaded only in the duodenum and liver, as in Hephint/int and Cp-/- mice, respectively. Both double knockout models absorbed iron in radiolabeled intestinal iron absorption studies, but the iron was inappropriately distributed, with an abnormally high percentage retained in the liver.ConclusionsThese studies indicate that HEPH and CP, and likely MCFs in general, are not essential for intestinal iron absorption but are required for proper systemic iron distribution. They also point to important extra-intestinal roles for HEPH in maintaining whole-body iron homeostasis.

Expression of Duodenal Iron Transporter Proteins in Diabetic Patients with and without Iron Deficiency Anemia.

The role of iron transport proteins in the pathogenesis of anemia in patients with diabetes mellitus (T2DM) is still unclear. We investigated the expression of duodenal transporter proteins in diabetic patients with and without iron deficiency anemia (IDA). Methods. Overall, 39 patients were included: 16 with T2DM and IDA (group A), 11 with T2DM without IDA (group B), and 12 controls (group C). Duodenal mucosal expression of divalent metal transporter 1 (DMT1), ferroportin 1 (FPN), hephaestin (HEPH), and transferrin receptor 1 (TfR) was evaluated by Western blotting. Chronic disease activity markers were measured as well. Results. FPN expression was increased in group A compared to group B and controls: 1.17 (0.72–1.46), 0.76 (0.53–1.04), and 0.71 (0.64–0.86), respectively (p = 0.011). TfR levels were over expressed in groups A and B compared to controls: 0.39 (0.26–0.61), 0.36 (0.24–0.43), and 0.18 (0.16–0.24), respectively, (p = 0.004). The three groups did not differ significantly with regard to cellular HEPH and DMT1 expression. The normal CRP and serum ferritin levels, accompanied with normal FPN among diabetic patients without IDA, do not support the association of IDA with chronic inflammatory state. Conclusion. In patients with T2DM and IDA, duodenal iron transport protein expression might be dependent on body iron stores rather than by chronic inflammation or diabetes per se.

Abstract 4680: H3K9 methyltransferase G9a epigenetically regulates breast cancer cell iron homeostasis via repressing ferroxidase hephaestin expression, promotes breast cancer growth

Abstract G9a is a H3K9 methyltransferase with elevated expression observed in many types of human cancers, seems to be required for the maintenance of the malignant phenotype. However, the tumorigenic role of G9a in breast cancer is still far from clear. In addition, growing evidence revealed that the disruption of iron homeostasis may contribute to the development of breast cancer (BC). Pathways of iron acquisition, efflux, storage and regulation are all perturbed in BC, suggesting that reprogramming of iron metabolism is a central aspect of tumor cells survival. Hephaestin (HEPH) is a ceruloplasmin homologue that converts iron (II) to iron (III), and mediates iron efflux in concert with ferroportin to transport iron across the basolateral membrane. It retains unclear whether HEPH concentration has any impact on the iron status of breast tissue, and breast cancer growth. In this study, we demonstrate that G9a exerts its oncogenic function by destruction cellular iron homeostasis. We revealed that G9a knockdown, and G9a specific inhibitors significantly up-regulated HEPH expression, decreased intracellular labile iron content, and then suppressed BC cells proliferation both in vitro and in vivo. This idea is further validated by the finding that the enhanced iron content and decreased HEPH expression are required for the increased proliferation of G9a-overexpressed breast cancer cells. Further, our CHIP and co-IP data demonstrate a traditional role of G9a as a transcriptional repressor that co-existed with YY1 and HDAC1, contribute to the reduction of HEPH transcription. In addition, depletion of HEPH with an increase in labile iron reversed cells growth in G9a silencing BC cells. These observations prompted us to investigate the relevance of G9a-HEPH pathway to human disease. The immunohistochemistry analysis of 75 breast cancer specimens revealed inverse staining patterns between G9a and HEPH expression in breast cancer tissues independent of BC type (Spearman's nonparametric correlation test, correlation coefficient: -0.678, p&amp;lt;0.05). Patients who owned a G9ahigh tumor had an even worse prognosis in this retrospective analysis, with a median overall survival of 56.9 months compared to 103.4 months in the G9alow tumors. And we revealed for the first time that low level of HEPH also correlated with overall lower disease-free survival. Finally, expression of high G9a and low HEPH correlated a worse prognosis. In summary, we propose that G9a has an upstream regulatory role in HEPH-mediated cellular iron hemostasis, leads to iron accumulation and stimulates breast cancer progression through its epigenetic silencing machinery. Our observations raise exciting possibilities regarding G9a and HEPH might be potential prognostic markers of breast cancer progression and targets for therapeutic intervention. Citation Format: Ya-fang Wang, Jie Zhang, Yi Su, Yan-yan Shen, Mei-yu Geng, Jian Ding, Yi Chen. H3K9 methyltransferase G9a epigenetically regulates breast cancer cell iron homeostasis via repressing ferroxidase hephaestin expression, promotes breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4680. doi:10.1158/1538-7445.AM2017-4680

Effects of Octylphenol and Bisphenol A on the Metal Cation Transporter Channels of Mouse Placentas.

Octylphenol (OP) and bisphenol A (BPA) are known as endocrine-disrupting chemicals (EDCs). During pregnancy, the expression of steroid hormone receptors is controlled by maternal and fetal nutrition. To evaluate the impact of EDCs during pregnancy, ethinyl estradiol (EE, 0.2 mg/kg/day), OP (50 mg/kg/day), and BPA (50 mg/kg/day) were administered to pregnant mice. The mRNA levels of TRPV6 (transient receptor potential cation channels in subfamily V, member 6) decreased significantly by EE and OP. The PMCA1 (ATPase, Ca++ transporting, plasma membrane 1) mRNA and protein levels decreased significantly by EE, OP, and BPA. CTR1 (solute carrier family 31, member 1) and ATP7A (ATPase, Cu++ transporting, alpha polypeptide) expression decreased significantly by EE, OP, and BPA. The mRNA levels of IREG1 (iron-regulated transporter, member 1) decreased significantly by EE. Hephaestin (HEPH) mRNA levels decreased significantly by EE, OP, and BPA, and protein levels decreased significantly by BPA. As a result of immunohistochemistry analysis, all cation transporter proteins were found in labyrinth of placenta. To confirm the cytosolic level of cations, levels of cation level in fetal serum were measured. EE, OP, and BPA significantly reduced serum calcium and copper levels, and iron levels were reduced by BPA. Taken together, some EDCs, such as OP and BPA, could modulate the calcium, copper, and iron ion-transporting channels during pregnancy. The fetus relies on the mother for ionic transportation, and, therefore, pregnant women should avoid exposure to cation-channel-disrupting chemicals.