Induction of FPN1 transcription by MTF-1 reveals a role for ferroportin in transition metal efflux

Abstract

IntroductionFerroportin (Fpn) is the only known mammalian iron exporter and plays an essential role in the entry of iron into plasma (for review, see Wessling-Resnick 1 and Wrighting and Andrews 2 ). Fpn is most highly expressed in cells that play a major role in iron acquisition: duodenal enterocytes, macrophages, hepatocytes and syncytial trophoblasts. Fpn, however, can be expressed on a wide variety of other cell types in response to heme 3,4 or iron. [5][6][7] Iron can regulate Fpn expression through transcriptional and translational mechanisms. Ferroportin1 (FPN1), the gene that encodes Fpn, contains a 5Ј iron-responsive element (IRE), which places its translation under the control of iron regulatory proteins. It is thought that the increased expression of Fpn by iron is a response to cellular iron load, as increased cytosolic iron would lead to increased iron export.FPN1 mRNA levels are also increased when cells are exposed to transition metals such as zinc, manganese, cadmium, copper, and other transition metals. 6 In both J774 cells and Caco cells, the increase in Fpn expression resulted in increased iron export. What is unclear, however, is the physiologic function of transition metal-induced expression of Fpn. Iron export in response to increased transition metals may protect cells from transition metal toxicity, perhaps by reducing the potential for iron-induced Fenton chemistry. Alternatively, Fpn might export other metals in addition to iron and thus increased expression of Fpn would directly protect cells from metal toxicity.In this study, we identify at least 1 mechanism by which transition metals induce Fpn expression. We show that zinc and cadmium can activate FPN1 transcription through the Metal Transcription Factor-1 (MTF-1). We also demonstrate that Fpn transports zinc and can protect cells from zinc toxicity. MethodsVector pcDNA3.1-mouse-MTF1-Flag was a gift from Dr G. K. Andrews. 8 We produced a chimera pcDNA3.1-mouse/human-MTF1-Flag by switching the mouse fragment with the human corresponding fragment between KpnI/FseI sites of mouse MTF1-Flag. To express an shRNA against mouse MTF-1, we inserted a double strand oligonucleotide targeting the gtacttcgccaccgctgta sequence of mouse MTF-1 into BamHI and EcoRI sites of pSIREN-DNR-DSRed (Clontech) according to manufacturer's instructions. The mouse/human chimera MTF-1 is resistant to shRNA against mouse MTF-1. The pGL3-control vector (Promega) was modified to perform the luciferase assay. The SV40 promoter was removed by HindIII/NheI digestion and the mouse Fpn promoter (starting at Ϫ2378, Ϫ1154, and Ϫ623 from the TATAA box) was amplified by polymerase chain reaction (PCR) and inserted using same restriction sites. The 5ЈIRE in the FPN1 promoter was deleted by BamHI/SamI digestion as described. 9 The putative metal-responsive elements (MREs) in the Fpn promoter in pGL3 were mutagenized by PCR to produce TCCAGCA*GA*AT*CT*CG and TGGAAGAA*TT*CG*AG (the consensus sequence is underlined, *mutagenized base). Fpn-green fluorescent protein (GFP) was car...

Disordered signaling governing ferroportin transcription favors breast cancer growth

“…MTF-1 could bind to the FPN promoter through the induction of zinc and cadmium to transactivate FPN expression in mouse macrophages and other non-tumor cells [21]. Nrf2 was identified to induce FPN expression in mouse macrophages by binding to the Maf recognition elements (MARE)/antioxidant response elements (ARE) enhancer sequence on the FPN promoter [23,30].…”

“…However, our current understanding of the transcriptional regulation of FPN is still rather limited. Burgeoning studies have documented that HIF-2α, Nrf2 and MTF-1 transcriptionally modulate FPN expression in mouse macrophages and other non-tumor cells [21][22][23]. Other than these, the upstream signaling responsible for FPN's transcriptional regulation remains elusive, especially in tumor cells.…”

The role of lysosomes in iron metabolism and recycling

“…In the enterocyte, iron is liberated by heme oxygenase 1 (Hmox 1) (Dunn et al, 2007). Iron is then transported away from the basolateral membrane of the enterocyte and from macrophages, by ferroportin 1, which is expressed in all iron-exporting cells and plays a critical role in the regulation of iron-export to the bloodstream (Donovan et al, 2005, Troadec et al, 2010.…”

Estrogen contributes to regulating iron metabolism through governing ferroportin signaling via an estrogen response element

“…Additionally, FPN could also be regulated by hypoxia inducible factor 2 (HIF2α), nuclear factor erythroid (Nrf2), and metalregulatory transcription factor 1 (MTF-1) at the transcriptional level in macrophages [11,12,16]. However, little is known about the regulation of FPN under different physiological settings including natural alterations of endogenous steroid hormones, such as estrogen.…”