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Biochimica et Biophysica Acta 1842 (2014) 2298–2305

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Biochimica et Biophysica Acta

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The soluble form of HFE regulates mRNA expression in the duodenum through an endocytosis-dependent mechanism

Bruno Silva a, Joana Ferreira a,VeraSantosa, Cilénia Baldaia b, Fátima Serejo b, Paula Faustino a,⁎

a Departamento de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal b Departamento de Gastroenterologia e Hepatologia, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, Lisboa, Portugal

article info abstract

Article history: Dietary absorption regulation is one of the key steps for the maintenance of the body iron homeostasis. Received 6 December 2013 HFE expression undergoes a complex post-transcriptional alternative splicing mechanism through which Received in revised form 25 June 2014 two alternative transcripts are originated and translated to a soluble HFE protein isoform (sHFE). The first pur- Accepted 15 July 2014 pose of this study was to determine if sHFE transcript levels respond to different iron conditions in duodenal Available online 27 July 2014 and macrophage cell models. In addition, we aimed to determine the functional effect of the sHFE protein on

Keywords: the expression of iron -related in a duodenal cell model as well as, in vivo, in duodenum biopsy Alternative splicing samples. Iron metabolism Levels of sHFE transcripts were measured in HuTu-80, Caco-2, HT-29 and activated THP1 cells, after holo-Tf stimulus, and in total RNA from duodenum biopsies of functional dyspepsia patients. Also, the expression of Iron absorption several iron metabolism-related genes was determined after endogenous and exogenous overexpression of Macrophage sHFE protein in a duodenal cell model. sHFE endocytosis mechanism was studied using endocytosis inhibitors. Endocytosis Our results showed that sHFE transcript expression was up-regulated after holo-Tf stimuli. Hephaestin and duo- denal cytochrome b expressions were down-regulated by both endogenous HFE and sHFE in a duodenal cell model. Exogenous sHFE was able to down-regulate hephaestin mRNA levels by a clathrin-independent, dynamin-mediated, and RhoA-regulated endocytosis mechanism. Moreover, HEPH levels negatively correlated with sHFE levels in the duodenum of functional dyspepsia patients. Thus, sHFE seems to be an important iron metabolism regulator playing a role in the control of dietary iron absorption in the duodenum. © 2014 Elsevier B.V. All rights reserved.

1. Introduction hephaestin (Heph, a membrane associated ferrous oxidase) [4,5], which also mediates iron efflux towards circulatory most Every day, the human body needs to absorb approximately 1 mg of likely in cooperation with the basolateral iron transporter, iron from the diet in order to compensate unspecific losses that may (Fpn1). occur [1]. As there are no active mechanisms to excrete iron, and since The liver is so far the main organ considered to be involved in iron both iron deficiency and overload can be harmful [1], the human body homeostasis regulation. As iron levels increase, there is concomitant en- has developed fine-tuned iron homeostasis regulation mechanisms. hancing in the synthesis of the hormone (coded by the HAMP These encompass iron absorption by duodenum , storage gene) by hepatocytes [6]. When released into the circulation, hepcidin in liver hepatocytes and recycling by macrophages. Regarding absorp- acts by binding to the membrane Fpn1, triggering its internalization tion, dietary iron is presented as heme or as Fe3+. The latter needs to and degradation. Therefore, it prevents the release of iron from cells, in- be reduced by the reductase (Cybrd1) at the cluding the dietary iron absorbed by the enterocytes [7]. The molecular enterocyte apical membrane, in order to be transported into the cell mechanisms responsible for HAMP gene expression are mediated via divalent-metal transporter 1 (Dmt1) [2,3]. Once in the cell, Fe2+ is through membrane-surface proteins such as HFE, transferrin recep- directed to the basolateral membrane where it is converted in Fe3+ by tors-1 and -2 (TfR1, TfR2), (Hjv) and bone morphogenetic protein receptors (BmpRs) [8,9].

Abbreviations: sHFE, soluble HFE; holo-Tf, iron-loaded transferrin; Cybrd1, duodenal HFE is a major histocompatibility complex class I-like protein which cytochrome b; Heph, hephaestin; Fpn1, ferroportin-1; TfR, ; TfSat, gene is commonly mutated in hereditary hemochromatosis (HH; OMIM β transferrin saturation; 2M, beta 2-microglobulin; PMA, phorbol 12-myristate 13- 235200), an autosomal recessive disorder characterized by excessive in- acetate; Dyn, dynasore; CPZ, chlorpromazine. testinal iron absorption and deposition in several organs [10]. The ex- ⁎ Corresponding author at: Departamento de Genética Humana, Instituto Nacional de pression of HFE undergoes a complex post-transcriptional regulation Saúde Dr. Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisboa, Portugal. Tel.: +351 217508164; fax: +351 217526410. that involves alternative polyadenylation and splicing mechanisms. So E-mail address: [email protected] (P. Faustino). far, there were identified four different polyadenylation signals and

http://dx.doi.org/10.1016/j.bbadis.2014.07.017 0925-4439/© 2014 Elsevier B.V. All rights reserved. B. Silva et al. / Biochimica et Biophysica Acta 1842 (2014) 2298–2305 2299 more than ten transcripts resulting from HFE alternative splicing in DMEM GlutaMAX™ medium (Invitrogen, Carlsbad, CA, USA) supple- [11–13]. The expression of these variants seems to be ubiquitous and mented with 10% (v/v) fetal bovine serum (FBS), while THP-1 cells their functional characterization is not yet complete. The full-length (human monocyte-derived cell line), Caco-2 and HT-29 cells HFE protein, resulting from the complete inclusion of the first six (human epithelial colorectal adenocarcinoma cells) (DSMZ, exons of HFE, is known to suffer intracellular processing through Germany) were maintained in RPMI 1640 medium (Invitrogen, which is associated with the chaperone β2-microglobulin (β2M) [14]. Carlsbad, CA, USA) supplemented with 10% (v/v) FBS, in a 37 °C/5% It is then transported to the cell surface, where it interacts with TfR1 CO2 incubator. in the presence of normal circulatory holo-transferrin (holo-Tf) levels For experiments, HuTu-80, THP-1 and HT-29 cells [15,16]. The main function of HFE is assumed as being the regulation were seeded in 35 mm plates at a confluence of 1 × 106 cells per well, of HAMP expression in the liver. It has been hypothesized that when while Caco-2 cells were seeded at a confluence of 5 × 105 cells holo-Tf levels increase, HFE dissociates from TfR1 and becomes available per well. THP-1 cells were seeded in the presence of 5 ng/mL phorbol to interact with TfR2 and Hjv, triggering signaling pathways that pro- 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA) for differen- mote HAMP gene expression by the hepatocytes [8,17–19]. However, tiation. Twenty-four hours after seeding (or 48 h for THP-1 and HT-29 this was recently challenged with results showing that HFE and TfR2 cells), the media were replaced with fresh media containing 20 μM do not interact and that the HFE-mediated HAMP expression activation holo-Tf or 20 μM apo-Tf (Sigma, St. Louis, MO, USA) (or the same does not require TfR2 [20,21]. On the other hand, it was also proposed volume of vehicle solution, H2O). Cells were harvested 12 h after that full-length HFE may act directly in enterocytes, regulating iron ab- stimulus was given. sorption. It was shown that HFE overexpression in colon colorectal ade- For transient transfection experiments, cells were seeded in 35 mm nocarcinoma HT-29 cell line inhibits iron efflux from cells possibly by plates at a confluence of 1 × 106 cells per well (or 5 × 105 cells for the down-regulation of HEPH expression [22]. Also, the expression of Caco-2). Twenty-four hours after seeding, 2 μgofthepcDNA+HFE CYBRD1, as well as the integrity of duodenal epithelium, was shown to constructs were used together with Lipofectamine 2000 Transfection be altered in HFE knockout mice [23–25]. Reagent (Invitrogen, Carlsbad, CA, USA) following the Amongst the HFE mRNA alternative splicing variants, two were manufacturer's instructions. Two hours after transfection medium shown to give rise to a truncated soluble HFE protein isoform (sHFE). was replaced with fresh medium containing 20 μM holo-Tf (or the

These alternative transcripts result from the total inclusion of HFE intron same volume of vehicle solution, H2O). Twenty-four hours later, 4 or the inclusion of the first 66 base-pairs of intron 4 and, in both cases, cell culture supernatant and cells were collected for immunoprecip- an in frame premature termination codon is formed, six nucleotides itation and RNA extraction. from the exon 4/intron 4 boundary [12]. The sHFE protein preserves The exogenous sHFE experiments were performed by producing an intact α3 domain but lacks the transmembrane domain and the cyto- sHFE protein in HEK293 cells. Briefly, HEK293 cells were seeded and plasmic tail. Consequently, it maintains the ability to associate with β2M transfected with 2 μg of the pcDNA + HFE constructs. Two hours later but not with TfR1 and it has been shown to be secreted to the extracel- the medium was replaced and after 48 h of culture, the supernatant lular environment in several cell types [12]. However, the function of was collected and centrifuged for clearing from dead cells and debris. sHFE is still unknown. The cleared supernatant was then added to previously seeded HuTu-80 In this study, we investigated if iron affects the expression levels of cells in a ratio of 3:1 (supernatant: fresh medium). Endocytosis inhibi- sHFE transcripts in duodenal and macrophage cell line models. We tion was carried on HuTu-80 cells by replacing the medium with fresh have also determined the effect of the presence of both the endogenous medium containing 40 μM dynasore (Sigma, St. Louis, MO, USA) or and exogenous sHFE isoform on the transcriptional regulation of several 10 μg/mL Chlorpromazine (CPZ; Sigma, St. Louis, MO, USA) 30 min be- iron metabolism-related genes in a duodenal cell line model. The results fore and when conditioned supernatant was added (or an equivalent obtained were validated by in vivo studies performed in duodenum volume of vehicle solution, DMSO). Cells were then harvested for RNA biopsies. 4 h later. For RhoA inhibition experiments HuTu-80 cells were transfected with 2 μg of pGFP (empty-vector) or pGFP-RhoA_N19, 24 h 2. Methods prior to the replacement with conditioned medium. Cells were then harvested for RNA and protein 4 h later. 2.1. Plasmid constructs 2.3. Patients' selection, peripheral blood biomarkers and isolation of duode- The cloning of both HFE and sHFE cDNAs into the pcDNA3 vector num biopsies (Clontech, Mountain View, CA, USA) took advantage of the constructs described in Martins and Silva et al., 2011 [12]. These constructs, contain- A group of 6 individuals referred for endoscopy due to dyspepsia ing the total HFE or the sHFE cDNAs, were used to amplify the entire cod- symptoms at the Departamento de Gastroenterologia e de Hepatologia, ing sequences with the forward primer 5′-ttttggtaccatgggcccgcgagcc-3′ Hospital Santa Maria, Lisbon, was enrolled in this study. Besides containing the KpnI linker and the reverse primers 5′-ttttgatatctcaga dyspepsia, these patients presented no other pathological conditions. tcttatcgtcgtcatccttgtaatccatctcacgttcagctaagacgtagtgccc-3′ or 5′-ttttga The ethical committee of the Hospital Santa Maria approved the study, tatctcagatcttatcgtcgtcatccttgtaatccatcataccccagatcacaatgagggg-3′ (for and all patients signed an informed consent. total HFE and sHFE, respectively) containing the EcoRV linker next to Patient's peripheral blood samples were obtained by venipuncture. the stop codons and Flag epitope sequence (stop codon was modified Serum was isolated and used for the measurement of iron metabolism in order to allow fusion to Flag open reading frame). Both pcDNA3 biomarkers by the standard protocols (Table 1). Duodenum biopsies empty vector and PCR products were digested with the KpnIandEcoRV were collected and immediately stored in RNA later (Quiagen, CA, endonucleases and ligation was performed in order to obtain the USA) at −80 °C. pcDNA + HFE-Flag and pcDNA + sHFE-Flag constructs. The sequences of constructs were confirmed by automated Sanger sequencing. The pGFP +RhoA_N19 construct was kindly given by Dr. Peter Jordan [26]. 2.4. RNA extraction and real time-qPCR

2.2. Cell culture and transfection Total RNA extraction from cultured cells was carried out by using a NucleoSpin® RNA II kit (Macherey Nagel, Düren, Germany) according HuTu-80 cells (human duodenal enterocyte cell line) and HEK293 to the manufacturer's instructions. RNA extraction from dyspepsia cells (used for protein production) (DSMZ, Germany) were maintained patients' duodenum biopsies was performed by a standard Trizol 2300 B. Silva et al. / Biochimica et Biophysica Acta 1842 (2014) 2298–2305

Table 1 2.7. Statistical analysis Characterization of iron status of the dyspepsia patients analyzed in the current work.

Patient Gender Age Serum iron Transferrin TfSat Serum Data are presented as means ± standard deviations obtained from (μg/dL) (μg/dL) (%) (μg/L) at least three independent experiments. Groups were compared with I F 49 80 270 29.6 41 Student's t test. Correlations were calculated with the PASW Statistics II F 21 70 359 19.4 201 20.0® software (SPSS Inc.). Two-sided p-values of b0.05 indicated III F 45 135 456 29.6 291 statistical significance, while one-sided p-values are presented for the IV M 52 91 327 27.8 825 control experiments. V M 34 145 400 36.3 50 VI M 51 120 282 42.6 300 3. Results M: male; F: female; and TfSat: transferrin saturation. 3.1. Holo-transferrin regulates the expression of sHFE transcripts in duodenal and macrophage cell models extraction protocol after tissue homogenization. RNA samples were treated with DNase I (Roche, Mannheim, Germany). In order to determine whether iron modulates the expression of the First strand cDNA synthesis was performed by using 2–5 μgoftotal alternative HFE transcripts responsible for the sHFE isoform production, RNA together with an equivalent mixture of random primers and we stimulated HuTu-80, Caco-2, HT-29 and activated THP-1 cells with oligo(dT)12–18 and the SuperScript II Reverse Transcriptase (Invitrogen, 20 μM holo-Tf for 12 h. After mRNA extraction, quantification of total Carlsbad, CA, USA), according to the manufacturer's instructions. To HFE, sHFE and TFR1 transcripts was performed (Fig. 1). We observed discard DNA contamination in the RNA samples a control PCR was that, in HuTu-80, Caco-2, HT-29 and activated THP-1 cells, TFR1 expres- performed for the ABL gene. sion was down-regulated by holo-Tf (0.65 ± 0.12, p = 0.0018; 0.57 ± Quantification of iron metabolism-related gene expression was con- 0.09, p = 0.0002; 0.61 ± 0.16, p = 0.0142 and 0.67 ± 0.08, p = ducted using real-time qPCR performed on an ABI Prism 7000 Sequence 0.0054, respectively). No significant differences were found in total Detection System. Primers were designed to amplify specific amplicons HFE expression (0.88 ± 0.10, p = 0.1330; 1.095 ± 0.21, p = 0.4548; of the total HFE, sHFE, TFR1, HAMP, IL1-β, SLC11A2, SLC40A1, CYBRD1, 1.042 ± 0.22, p = 0.7447 and 0.95 ± 0.24, p = 0.7263 for HuTu-80, HEPH and HPRT1 transcripts (Table S1). Each cDNA sample was diluted Caco-2, HT-20 and activated THP-1 cells, respectively). On the other 5-fold and then 5 μL of dilutions added to 5 pmol of each primer and hand, the expression of the sHFE transcripts increased with holo-Tf in SYBR Green Master Mix (Applied Biosystems, Warrington, UK). The HuTu-80, Caco-2 and activated THP-1 cells (1.37 ± 0.16, p = 0.0269; cycling parameters were: 10 min at 95 °C, followed by 40 cycles of 15 1.26 ± 0.10, p = 0.0241 and 1.54 ± 0.27, p = 0.0280, respectively) s at 95 °C and 1 min at 60 °C. Quantification of gene expression was per- (Fig. 1A, B and D), while no differences were found in the HT-29 cells formed by the 2^−ΔCt method using HPRT1 as the normaliser gene. Each (1.05 ± 0.18, p = 0.6714) (Fig. 1C). Also, the mRNA levels of TFR1, sample was quantified in triplicate and primer amplification efficiencies total HFE and sHFE do not vary with apo-Tf stimuli in Hutu-80 and were calculated and validated with the standard curves obtained Caco-2 cells (Fig. S1). The control of THP-1 activation by PMA was through the amplification of cDNA serial dilutions. performedbythequantification of IL1β mRNA levels (Fig. S2).

3.2. sHFE protein isoform overexpression down-regulates CYBRD1 and 2.5. Immunoprecipitation HEPH transcript expressions in Hutu-80 cells

Twenty-four hours after transfection, both cell media (b2 mL) and Since the previous experiments revealed increased sHFE transcript platted cells were harvested. Cell media were centrifuged and the su- levels in response to holo-Tf stimulus, we investigated the effect of the pernatants collected. A total of 150 μL of lysis buffer was then added, overexpression of both the full-length HFE and the sHFE proteins in on ice, to this supernatant and cells [12]. The supernatant and cell ly- Hutu-80, Caco-2 and HT-29 cells in the expression of several iron me- sates were cleared by centrifugation and an aliquot of 20 μL transferred tabolism genes (Figs. 2 and S3). The immunoblot results confirmed to 2× Laemmli buffer. Then 8 μL of mouse anti-Flag M2 antibody (Sigma, the transient overexpression of both full-length HFE- and sHFE-Flag fu- St. Louis, MO, USA) was added and after one hour of incubation at 4 °C, sions independently of holo-Tf in HuTu-80 cells (Fig. 2A). The immuno- 60 μL of G-agarose bead slurry was added overnight. The beads were precipitation performed on cell lysates showed that the HFE full-length spun down, washed three times and the protein was resuspended in protein was associated with β2M and TfR1 in normal conditions 2× Laemmli buffer. (Fig. 2B). However, when holo-Tf was added, we could no longer observe the association with TfR1 (Fig. 2B, left panels). On the other

hand, sHFE isoform was found associated only with β2M. Also, as 2.6. Western blot analysis expected, the immunoprecipitation performed on the cell culture supernatant showed that the full-length HFE was not present in the Proteins from cell lysates or from cell culture supernatants were extracellular environment. Conversely, we have detected sHFE protein resolved in a 12% SDS-PAGE and transferred to PVDF membranes associated with β2M, independently of iron status (Fig. 2B, right panels). (Bio-Rad, Hercules, CA, USA), which were blocked using a 10% (m/v) Regarding the effect of the overexpression of both HFE and sHFE TBST-Milk solution. Membranes were probed using rabbit anti-Flag proteins on iron metabolism gene expression in HuTu-80 cells, we did (Sigma, St. Louis, MO, USA) at a 1:500 dilution, rabbit anti-GFP not detect significant differences on the levels of TFR1, SLC11A2 and

(Abcam, Cambridge, UK) at a 1:5000 dilution, rabbit anti-β2M SLC40A1 (Fig. 2C–E). However we did observe a decrease of CYBRD1 (Abcam, Cambridge, UK) at a 1:500 dilution or mouse anti-TfR1 and HEPH gene expression (Fig. 2F and G). Particularly, the expression (Invitrogen, Carlsbad, CA, USA) at a 1:500 dilution. For pre-IP lysates, of CYBRD1 decreased approximately 20% with the overexpression of a mouse anti-HSP90β antibody (Invitrogen, Carlsbad, CA, USA) was both the full-length HFE and sHFE proteins (0.79 ± 0.07, p = 0.0418 used as a loading control at a 1:100 dilution. Detection was carried and 0.77 ± 0.04, p = 0.0190, respectively) (Fig. 2F). The observed out using secondary peroxidase-conjugated anti-mouse IgG (Bio-Rad, differences were shown to be independent of holo-Tf stimuli. On the Hercules, CA, USA) at a 1:4000 dilution or anti-rabbit IgG (Bio-Rad, Her- other hand, the overexpression of full-length HFE led to a 16% decrease cules, CA, USA) at a 1:3000 dilution, followed by chemiluminescence of HEPH expression (0.84 ± 0.04, p = 0.0302). This decrease was even assays. higher when holo-Tf was added (0.53 ± 0.12, p = 0.0083) (Fig. 2G). B. Silva et al. / Biochimica et Biophysica Acta 1842 (2014) 2298–2305 2301

Fig. 1. Effect of holo-Tf on the expression of sHFE transcripts. (A) HuTu-80, (B) Caco-2, (C) HT-29 and (D) PMA-activated THP-1 cells were stimulated with medium containing 20 μM holo-Tf for 12 h and the expression of TFR1, total HFE and sHFE was analyzed by RT-qPCR. Mock refers to the control cells without the stimuli but with the same amount of vehicle solution (H2O). Values were obtained by normalizing first the expression to HPRT1 and then to the mock control. Bars represent the standard deviations from at least three independent experiments; *p b 0.05; **p b 0.001.

Similarly, the overexpression of sHFE caused a decrease of 31% on This result was, however, different when Caco-2 and HT-29 HEPH's expression (0.69 ± 0.14, p = 0.0235) (Fig. 2G). Under holo-Tf cells were transfected with the same constructs, as no signifi- stimulus the expression decreased to approximately half of the normal cant differences in CYBRD1 and HEPH expressions were found expression (0.47 ± 0.04, p = 0.0001) (Fig. 2G). (Fig. S3).

Fig. 2. Effect of sHFE protein overexpression on the levels of expression of iron metabolism-related genes in HuTu-80 cells. HuTu-80 cells were transfected with pcDNA3 empty vector (mock), pcDNA + HFE-Flag and pcDNA + sHFE-Flag constructs under regular and iron overload conditions (−holo-Tf and +holo-Tf, 20 μM, respectively). Twenty-four hours later, cells were harvested for RNA and protein and culture supernatant collected. Protein lysates and cell culture supernatant were subjected to immunoprecipitation using a mouse anti- Flag monoclonal antibody and G-agarose beads. Immunoblot detection was performed on (A) crude cell lysates and (B) immunoprecipitation products from cells (left panels) and cell culture supernatants (right panels). Blots were incubated with anti-Flag, -TfR1, -β2M and -HSP90β antibodies for protein detection. RNAs were used to assess the expression levels of (C) TFR1 (D) SLC11A2 (E) SLC40A1 (F) CYBRD1 and (G) HEPH by RT-qPCR. Values were obtained by normalizing first the expression to HPRT1 and then to the mock control without holo-Tf. Bars represent the standard deviations from at least three independent experiments; *p b 0.05; **p b 0.001. 2302 B. Silva et al. / Biochimica et Biophysica Acta 1842 (2014) 2298–2305

3.3. HEPH transcript expression is down-regulated by exogenous sHFE HEPH expression by exogenous sHFE (0.57 ± 0.21, p = 0.0271) protein isoform through an endocytosis dependent mechanism (Fig. 3E), while it partially prevented the TFR1 down-regulation effect by holo-Tf (1.01 ± 018, p = 0.2259) (Fig. 3F). The expression of transcripts that can give rise to the sHFE protein To investigate whether RhoA-regulated endocytosis is the mecha- was previously described as ubiquitous [12]. Here we have found that nism responsible for sHFE endocytosis, we transfected HuTu-80 the endogenous overexpression of this protein in HuTu-80 cells induces cells with a dominant negative form of RhoA GTPase (RhoA_N19) a decrease in the expression of CYBRD1 and HEPH transcripts. Therefore, prior to adding exogenous sHFE (Fig. 4). We found that the overexpres- we aimed to determine if the presence of the exogenous sHFE protein sion of RhoA_N19 inhibited HEPH down-regulation by exogenous sHFE could also induce the same effect on the expression of these genes in (p = 0.0322) (Fig. 4B). the same duodenal cell model. In order to achieve this, we transfected HEK293 cells with pcDNA + HFE and + sHFE constructs. Then, the con- 3.4. HEPH and sHFE expression levels are correlated in the human ditioned medium was used to stimulate HuTu-80 cells and the expres- duodenum sion of CYBRD1 and HEPH was measured. We observed that no effect was found in the expression of CYBRD1 four hours after the stimulus Since we found that the sHFE transcript expression levels increased (Fig. 3A). This expression remained unchanged even when the exposure with iron stimulus in duodenal and macrophage cell lines, we aimed to conditioned media was extended up to 24 h (data not shown). Oth- to determine if, in vivo, there is a correlation between individual iron erwise, we found that the exposure of these cells to sHFE conditioned status, evaluated by the peripheral blood iron metabolism biomarkers medium induced the decrease of HEPH expression (0.73 ± 0.19, p = (Table 1), and sHFE transcript expression in the duodenum (Fig. 5). 0.0309) (Fig. 3B). However, when we inhibited dynamin-dependent For this, we measured the mRNA levels of sHFE and HEPH in total endocytosis with dynasore, we no longer observed the same effect RNAs extracted from dyspepsia patients' duodenum biopsies. (1.04 ± 0.13, p = 0.5917) (Fig. 3B). The efficacy of endocytosis inhibi- We did not observe significant correlations between duodenal sHFE tion was tested by the effect of holo-Tf on TFR1 expression. As expected, expression and peripheral biomarker levels of iron status — serum iron, the observed decrease of TFR1 expression was significantly repressed in transferrin saturation (TfSat) and serum ferritin (Fig. 5A–C). Neverthe- the presence of dynasore (p = 0.0071) (Fig. 3C), therefore validating less, we observed a significant negative correlation between sHFE and dynamin-dependent endocytosis inhibition. The inhibitory effect of ex- HEPH expressions in the human duodenum (p =0.023)(Fig. 5D). ogenous sHFE in the expression of HEPH was not observed in either Caco-2 or HT-29 cells (Fig. S4). 4. Discussion Since the dynamin-dependent endocytosis mechanism may be, or not, clathrin-dependent, we further analyzed the effect of CPZ In order to access circulation, dietary iron has to surpass the barriers (clathrin-dependent endocytosis inhibitor) in HEPH down-regulation created at both the apical and basolateral membranes of the enterocyte. by exogenous sHFE in HuTu-80 cells. We observed that, the treatment The first step consists in the luminal reduction of Fe3+ by Cybrd1 and of HuTu-80 cells with CPZ did not prevent the down-regulation of transport into the enterocyte by Dmt1 [2,3].Secondly,ironcrossesthe

Fig. 3. Effect of exogenous sHFE stimulus on the expression of iron metabolism-related genes in HuTu-80 cells. HEK293 cells were transfected with pcDNA emptyvector(mock),pcDNA+HFE- Flag and pcDNA + sHFE-Flag constructs. Forty-eight hours post-transfection cell culture supernatants were collected and then used to stimulate previously cultured HuTu-80 cells. Endocytosis was inhibited with dynasore 40 μM (dyn) or chlorpromazine 10 μg/mL (CPZ). Four hours later, cells were harvested for RNA and the expression of (A, D) CYBRD1 and (B, E) HEPH was evaluated by RT-qPCR. (C, F) HuTu-80 cells were stimulated with medium containing both holo-Tf 20 μM and dynasore 40 μM or chlorpromazine 10 μg/mL, to control the inhibition of endocytosis mea- sured as the inhibition of TFR1 decrease by holo-Tf. Values were obtained by normalizing first the target gene expression to HPRT1 and then to the mock control. Bars represent the standard deviations from at least three independent experiments; *p b 0.05; **p b 0.001. B. Silva et al. / Biochimica et Biophysica Acta 1842 (2014) 2298–2305 2303

basolateral membrane in a process mediated by Fpn1 together with Heph, the enterocyte membrane-bound ferrous oxidase [4]. Whenever iron fails to cross this second barrier it remains inside the enterocytes and is eliminated by cell desquamation. Also, it is known that when body iron levels rise, the expression of hepcidin is stimulated in the liver, secreted into the circulation and, consequently, Fpn1 is internal- ized and degraded, therefore preventing iron absorption [7].Oneof the mechanisms underlying HAMP expression regulation in the liver in response to iron is co-dependent on the presence of HFE protein in the hepatocyte membrane. Regardless, HFE has a ubiquitous expression in human cells and it may also act directly in the enterocyte preventing iron absorption [22].Inaddition,HFE gene expression undergoes com- plex post-transcriptional processing and, to date, several alternative splicing transcripts have been described. Of these, two, originated from the total or partial intron 4 inclusions, give rise to the soluble form of HFE protein [12]. Previous studies have shown that HFE mRNA levels do not vary as a consequence of iron overload [27,28]. Accordingly, in this study we ob- served that total HFE mRNA levels remain unchanged upon holo-Tf stimulus in duodenal and activated-monocyte cell lines. However, we found that the level of the transcripts which originate the sHFE protein isoform increases with holo-Tf but not with apo-Tf stimulus. Therefore, we hypothesize that holo-Tf may somehow up-regulate the expression of HFE intron 4 inclusion variants while down-regulating other alterna- tive splicing variants and therefore maintaining the levels of total HFE expression. Actually, it has been reported that iron modulates aberrant Fig. 4. Effect of RhoA-mediated endocytosis inhibition in HEPH downregulation by exoge- splicing of ferrochelatase by acting as co-factor for the lysyl- nous sHFE in HuTu-80 cells. HuTu-80 cells were transfected with pGFP empty-vector hydroxylation of the splicing factor U2AF, important for the binding to (GFP) or with pGFP + RhoA_N19, 24 h prior to medium replacement with sHFE the polypyrimidine tract upstream from the 3′ splice site [29].Also, conditioned medium. Cells were then harvested after 4 h for protein and RNA. the expression of SFRS5 gene, coding for the serine–arginine-rich phos- (A) Immunoblot detection was performed on protein lysates and blots were incubated with anti-GFP and -HSP90β antibodies for protein detection. (B) HEPH expression was phoprotein 40 (SRp40), an important regulator of pre-mRNA splicing, evaluated by RT-qPCR. Values were obtained by normalizing first the target gene expres- was found up-regulated in the duodenum of mice fed with an iron- sion to HPRT1 and then to the mock control. Bars represent the standard deviations from at supplemented diet [30]. Indeed, in the HFE intron 4 sequence there least three independent experiments; *p b 0.05; **p b 0.001. are six motifs with affinity for the SRp40 (data not shown) and it was

Fig. 5. Correlation of duodenal sHFE expression levels with peripheral blood iron metabolism biomarkers and duodenal HEPH expression. Duodenum biopsies from dyspepsia patients were used for RNA extraction and the expression levels of sHFE and HEPH transcripts assessed by RT-qPCR using HPRT1 as a normalizer gene. Correlations were established between sHFE and (A) serum iron, (B) Tf sat, (C) serum ferritin and (D) HEPH expression. Trend lines are presented; r represents the Pearson's correlation value. 2304 B. Silva et al. / Biochimica et Biophysica Acta 1842 (2014) 2298–2305 shown that an increased expression of SRp40 is associated with large that iron may regulate sHFE expression, as the individuals here analyzed CD44 inclusion variants as well as with the translation of unspliced did not have abnormal peripheral blood iron parameter values. It would HIV type 1 RNA [31,32]. So, we hypothesize that the increased expres- be important to perform these experiments in groups of patients sion of sHFE observed in the presence of holo-Tf may be due to an in- presenting different iron status profiles. crease in SRp40 that will mediate the HFE intron 4 inclusion. Taken together, our results show that holo-Tf up-regulates the ex- Heph is a membrane associated ferrous oxidase that is responsible pression of the sHFE. Also, we unveiled a role for the sHFE in the regula- for iron oxidation whenever it is exported by Fpn1 through the tion of enterocyte HEPH expression that may affect cellular iron efflux basolateral membrane of enterocytes. It is preferentially expressed in and consequently dietary iron absorption. Vujic-Spasic and co- these cells, but it seems to be also expressed in the antrum, enteric ner- workers have shown that the ablation of HFE in the intestine of mice vous system and pancreas [33]. So far, little is known about its expres- did not affect the physiological control of systemic iron homeostasis sion regulation. It has already been reported that HEPH levels are not [44], concluding that HFE would not have a role in the intestine under affected in HH patients [34]. However, other authors observed that its steady state conditions. However, since sHFE is a putative circulatory expression was down-regulated after transfecting HFE in HT-29 cells protein, we can hypothesize that its expression in the intestine might [22]. Here, we also showed that the overexpression of HFE in HuTu-80 not be essential as other tissues may be overexpressing this isoform, cells down-regulated HEPH, suggesting a role of HFE in the regulation as seen in this report with the activated-THP1. We propose that an in- of HEPH expression. However, when we used HT-29 cells we were not crease on transferrin saturation may drive to the increase of sHFE ex- able to reproduce the same effect. Probably, this difference can be justi- pression in several tissues. Then sHFE will act by down-regulating fied by the location of the primer sets used for quantification, which be- HEPH expression and, most likely, inhibiting iron export from sides amplifying the major variant of HEPH transcript may also amplify enterocytes preventing the increase of body iron levels to a higher other HEPH alternative transcripts. extent. A similar down-regulatory effect on HEPH expression was also found Since the regulation of iron absorption is a key-step for the mainte- consequently to the overexpression of the sHFE protein. Since sHFE does nance of body iron levels, sHFE may be considered as a potential target not have the transmembrane and cytoplasmic domains, we can con- to the development of new therapeutic strategies for both iron deficien- clude that these protein domains are not involved in HEPH regulation. cy and overload pathologies. Also, it is known that sHFE is a circulatory protein, and that it might Supplementary data to this article can be found online at http://dx. be expressed by several tissues [12]. Our results proved that exogenous doi.org/10.1016/j.bbadis.2014.07.017. sHFE is able to down-regulate HEPH through a clathrin-independent, dynamin-mediated and RhoA-regulated endocytosis mechanism. Financial support Endocytosis is an important cellular process responsible for the inter- nalization of a diversity of molecules. Functionally it may work as a This study was partially supported by Fundação para a Ciência e a mechanism for nutrient acquisition, damaged cell surface repairing or Tecnologia (FCT) — grants PTDC/SAU-GMG/64494/2006 and PTDC/ even a mechanism mediating cell surface signaling pathways [35].It SAU-GMG/103307/2008, CIGMH and Pest-OE/SAU/UI00009/2011. BS has been reported that Rho-A regulated endocytosis is dependent on re- was supported by a FCT fellowship — SFRH/BD/60718/2009. ceptors located in lipid rafts for vesicle formation. For instance, interleukin-2 receptor has been shown to be endocytosed by Rho-A reg- fl ulated mechanism after binding to its ligands, promoting T cell prolifer- Con ict of interest ation [36]. Also, the Clostridium botulinum C2 toxin is internalized by a fl similar pathway [37]. Since several cell surface molecules related to The authors declare no con ict of interest. iron-metabolism regulation are located in lipid rafts, we hypothesize that the sHFE binds to one of those proteins, triggering a signaling Acknowledgements pathway responsible for HEPH expression down-regulation. Besides Heph, (Cp) is other known ferrous oxidase The authors thank Unidade de Tecnologia e Inovação, DGH, INSA, for expressed in the human body. It exists both in circulation and attached technical support. We also thank Drs. François Cannone-Hergaux, Peter to the cell membrane of hepatocytes and of peripheral blood mononu- Jordan, and Isabel Rivera for kindly providing THP-1, HEK293, Caco-2 clear cells [38]. In the current work we also have analyzed the effect of and HT-29 cells, and CPZ. Finally, we thank Dr. João Lavinha for the exogenous sHFE in the expression of CP in activated THP-1 cells. Howev- fruitful discussions concerning the current work. er, we observed no changes on its expression (data not shown). Despite playing a role in iron oxidation and transport, Cp and Heph have References distinct expression patterns and regulatory mechanisms [39]. In addi- tion, Heph is not able to oxidize adrenaline and dopamine as Cp does [1] M.B. Zimmermann, R.F. Hurrell, Nutritional iron deficiency, Lancet 370 (2007) [40]. So, it is not surprising that sHFE is not involved in the regulation 511–520. [2] H. Gunshin, B. Mackenzie, U.V. Berger, Y. Gunshin, M.F. Romero, W.F. Boron, S. of Cp expression. 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