Rafał R Starzyński

François Canonne-Hergaux

Alexandra Willemetz

Mikołaj A Gralak

Jarosław Woliński

Agnieszka Styś

Jarosław Olszak

Paweł Lipiński
email: p.lipinski@ighz.pl

Hemolytic anemia and alterations in hepatic iron metabolism in aged mice lacking Cu,Zn-superoxide dismutase

Keywords: erythrophagocytosis, iron, hemolysis, heme oxygenase, Kupffer cells, superoxide dismutase 1, Biochemical

) 402, [219][220][221][222][223][224][225][226][227] the chronic but moderate regenerative hemolytic anemia of old superoxide dismutase 1 (SOD1) knockout mice is associated with red blood cells modifications and sensitivity to both intra-and extravascular hemolysis. We have then characterized the molecular pathways of iron turnover in the liver of SOD1-deficient mice. Despite iron accumulation in liver macrophages namely Kupffer cells, we did not measure any significant change in non-heme liver iron. Interestingly in Kupffer cells, expression of the rate-limiting enzyme in heme degradation, the heme oxygenase-1 and expression of the iron exporter ferroportin were both upregulated whereas hepcidin mRNA level in the liver was decreased in SOD1 -/-mice. These results suggest that concerted changes in the hepatic expression of iron-and heme-related genes in response to hemolytic anemia in SOD1 -/-mice act to reduce toxic iron accumulation in the liver and respond to the needs of erythropoiesis.

INTRODUCTION

Reactive oxygen species (ROS) are by-products of normal aerobic metabolism. Several antioxidant systems exist in cells to provide protection against ROS toxicity. The principal defense against the toxicity of superoxide (O 2

.-

), released mainly into the cytosol [START_REF] Okado-Matsumoto | Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria[END_REF], is superoxide dismutase 1 (SOD1, Cu,Zn-SOD). Surprisingly, despite this crucial protective role, homozygous mutant mice lacking SOD1 are viable and phenotypically normal [START_REF] Reaume | Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury[END_REF][START_REF] Matzuk | Ovarian function in superoxide dismutase 1 and 2 knockout mice[END_REF][START_REF] Sentman | Phenotypes of mice lacking extracellular superoxide dismutase and copper-and zinc-containing superoxide dismutase[END_REF].

Permanent oxidative stress occurring due to elevated ROS later in life is an important mechanism in the pathogenesis of ageing [START_REF] Rattan | Theories of biological aging: genes, proteins, and free radicals[END_REF]. In older mice, SOD1 deficiency results in an increased incidence of pathological changes [START_REF] Elchuri | CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life[END_REF][START_REF] Keithley | Cu/Zn superoxide dismutase and age-related hearing loss[END_REF][START_REF] Muller | Absence of CuZn superoxide dismutase leads to elevated oxidative stress and acceleration of age-dependent skeletal muscle atrophy[END_REF]. In consequence, the lifespan of KO SOD1 mice is reduced by about 30% [START_REF] Matzuk | Ovarian function in superoxide dismutase 1 and 2 knockout mice[END_REF][START_REF] Sentman | Phenotypes of mice lacking extracellular superoxide dismutase and copper-and zinc-containing superoxide dismutase[END_REF].

Mice with genetic SOD1 deficiency have been extensively used as an experimental animal model to explore SOD1 dysfunction-related pathologies [START_REF] Elchuri | CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life[END_REF][START_REF] Muller | Absence of CuZn superoxide dismutase leads to elevated oxidative stress and acceleration of age-dependent skeletal muscle atrophy[END_REF][START_REF] Baumbach | Hypertrophy of cerebral arterioles in mice deficient in expression of the gene for CuZn superoxide dismutase[END_REF]. With regard to hematological disorders, the critical role of alterations in redox state due to SOD1 deficiency has been reported in hemolytic anemia (HA) occurring in SOD1 -/-mice [START_REF] Iuchi | Elevated oxidative stress in erythrocytes due to an SOD1 deficiency causes and triggers autoantibody production[END_REF], as well as in the impairment of hematopoietic cell development in the mouse model of Fanconi anemia [START_REF] Hadjur | Defective hematopoiesis and hepatic steatosis in mice with combined deficiencies of the genes encoding Fancc and Cu/Zn superoxide dismutase[END_REF].

In this study we examined the consequences of HA in 1-year-old mice lacking Cu,Zn-SOD1 activity, focusing on alterations in their hepatic iron metabolism. Our data clearly indicate that the enhanced destruction and accelerated removal of RBC from the circulation of old SOD1 -/-mice induce substantial changes in the expression of genes involved in heme and iron homeostasis. Our results establish a new link between oxidative stress due to the combined effects of SOD1 deficiency and age-dependent changes in redox state and the regulation of iron metabolism at the systemic level.

EXPERIMENTAL Animals

Mice homozygous for the non-functional SOD1 allele, and control mice homozygous for the wild-type SOD1 allele (wt) were used in experiments at the age of 1 year. A breeder pair of mice (strain B6;129S7-SOD1 tm1Leb ) heterozygous for a SOD1 tm1Leb targeted mutation [START_REF] Matzuk | Ovarian function in superoxide dismutase 1 and 2 knockout mice[END_REF] were provided by The Jackson Laboratory (Bar Harbor, ME). The 3rd Local Ethical Commission approved all experimental procedures involving animals. (permission no. 46/2006).

Analysis of RBC and serum iron parameters

Blood was drawn from mice by direct cardiac puncture immediately after death and was treated with heparin. RBC and reticulocytes counts, and cell parameters as well as serum iron were determined using an automated Sysmex F-820 Analyzer. Serum transferrin was measured at the Laboratory of Biochemistry at the Institut Federatif de Recherche 02, CHU Bichat-Claude Bernard (Paris, France).

Real-time quantitative RT (reverse transcriptase)-Polymerase Chain Reaction

Hepatic ferroportin, hepcidin, CD91, CD163 mRNA levels were measured by real-time quantitative PCR method as described previously [START_REF] Starzyński | Down-regulation of iron regulatory protein 1 activities and expression in superoxide dismutase 1 knock-out mice is not associated with alterations in iron metabolism[END_REF]. Real-time quantitative RT-PCR of cDNAs derived from specific transcripts was performed in a Light Cycler (Roche Diagnostics, Mannheim, Germany) using the respective pairs of oligonucleotide primers indicated in supplementary data (Table 1). For data analysis, Light Cycler 3.5 Software was used. Expression was quantified relative to that of a control transcript encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fifty micrograms of liver cytosolic extracts (for ferritin (Ft) subunit detection) or 100 µg of liver membrane extracts [for heme oxygenase-1 (HO-1) and ferroportin (Fpn) detection] were resolved on 14% and 10% SDS-polyacrylamide gels, respectively. To determine plasma haptoglobin (Hp) and hemopexin (Hx) levels, samples were separated on 10% SDSpolyacrylamide gels. Resolved proteins were electroblotted onto Hybond-ECL nitrocellulose membranes (Amersham Life Sciences, Little Chalfont, UK). The membranes were initially blocked by gentle agitation in TBST (0.15% Tween 20 in Tris-buffered saline) containing 5% fat-free powdered milk for 1 hour at room temperature (RT) followed by overnight incubation at 4°C with primary antibodies: rabbit antisera raised against recombinant mouse H-and Lferritin (H-and L-Ft; kindly provided by Dr. P. Santambrogio, Milan, Italy), rabbit polyclonal antibody raised against rat liver HO-1 (StressGen, Canada), rabbit antibody raised against mouse Fpn [START_REF] Canonne-Hergaux | Comparative studies of duodenal and macrophage ferroportin proteins[END_REF], chicken antibody raised against human Hp (USBiological, Swampscott, MA), and goat antiserum raised against human Hx (kindly provided by Dr. E. Tolosano, University of Turin, Italy). Membranes were then washed and incubated with peroxidaseconjugated anti-rabbit, anti-chicken or anti-goat secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour at room temperature. Immunoreactive bands were detected using the ECL Plus enhanced chemiluminescence Western blotting detection system (Amersham Life Sciences). Quantification was performed relative to actin detected using a specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Western Blot analysis

Immunohistofluorescence

Mouse livers were fixed in Bouin's for 72 hours at 20°C and then embedded in paraffin for sectioning. After mounting on glass slides, 5 µm sections were processed for immunofluorescence. The sections were first counterstained with 0.1% methylene blue in PBS and mounted in Permount. The sections were then incubated in a blocking solution for 45 minutes at RT. Incubation with primary antibodies was performed in a humid chamber at RT for 16 hours using the following dilutions in blocking solution: anti-Fpn, 1/75 and anti-HO-1, 1/100. After three washes with PBS/0.5% BSA, the sections were incubated for 1 hour at RT with a goat anti-rabbit-Alexa 488-conjugated secondary antibody (Molecular Probes) diluted 1/200 in blocking solution. After washing, the slides were coverslipped with antifading mounting reagent (Prolong Antifade kit P-7481, Molecular Probes) and examined for immunofluorescence.

Quantitative hepatic non-heme iron measurement and hepatic iron staining

Hepatic non-heme iron content was determined by acid digestion of liver samples at 65 o C for 20 hours followed by colorimetric measurement of the absorbance of iron-ferrozine complex at 560 nm as described previously [START_REF] Torrance | Iron stores[END_REF]. Deparaffinized liver sections were stained with Prussian blue and counterstained with nuclear red using standard procedures.

FACS analysis of phosphatidylserine (PS) externalization

The presence of PS on the surface of mouse RBC membranes was assessed using Annexin V-FITC staining (Calbiochem) following the manufacturer's protocol. Stained cells were analyzed by flow-cytometry (FACS Calibur), and data were processed using CellQuest software (Becton Dickinson).

Erythrophagocytosis assay (EP)

EP assay was performed as described previously [START_REF] Delaby | A physiological model to study iron recycling in macrophages[END_REF] using bone marrow-derived macrophages (BMDM) isolated from the femurs of SOD1 -/+ mice and fresh RBCs obtained from 1-year-old mice of both SOD1 -/-and SOD1 +/+ genotypes. A c c e p t e d M a n u s c r i p t

Heme content measurement

The heme content of formic acid-solubilized BMDM was determined spectrophotometrically at 398 nm using hemin standards prepared in formic acid and an extinction coefficient of 1.5×10 5 M 1 cm 1 [START_REF] Motterlini | Oxidative-stress response in vascular endothelial cells exposed to a cellular hemoglobin solutions[END_REF].

Statistical evaluation

Where suitable, one-way analysis of variance was applied to the data. The Scheffe test was used to estimate the significance of difference (p<0.05) between means. All calculations were performed using Statgraphics Plus 6.0 software.

RESULTS

Moderate regenerative anemia in old SOD1 -/-mice 1-year-old SOD1 -/-mice show moderate anemia with reductions of about 25% in both erythrocyte number and hemoglobin (Hb) concentration, and a reduction of about 14% in the hematocrit value (Table 1). Values for the mean cell Hb concentration were unchanged, which together with normal levels of serum iron (

Hemoglobin and heme scavenging systems in SOD1 -/-mice

The findings obtained by Iuchi et al. [START_REF] Iuchi | Elevated oxidative stress in erythrocytes due to an SOD1 deficiency causes and triggers autoantibody production[END_REF] indicate that SOD1 -/-RBC are prone to hemolysis due to oxidative damage. Therefore, we attempted first to identify hallmarks of intravascular hemolysis. We measured plasma levels of haptoglobin (Hp) and hemopexin (Hx), two glycoproteins involved in the clearance of Hb and heme, respectively, from the blood stream. When Hb and hem are released from ruptured erythrocytes they are instantly bound by Hp and Hx and form Hp-Hb and Hx-heme high affinity complexes [START_REF] Ascenzi | Hemoglobin and heme scavenging[END_REF]. These complexes are then rapidly taken up from circulation by CD163 [START_REF] Kristiansen | Identification of the hemoglobin receptor[END_REF] and CD91 [START_REF] Hvidberg | Identification of the receptor scavenging hemopexin-heme complexes[END_REF] receptors present on tissue macrophages and hepatocytes. The rationale for measuring Hp and Hx in the plasma is that CD163 and CD91 receptors have no measurable affinity for free Hp or Hx. Thus, specific recognition of Hp-Hb and Hx-heme complexes by CD163 and CD91 explains the decrease in Hp or Hx concentration in the plasma during accelerated hemolysis [START_REF] Hvidberg | Identification of the receptor scavenging hemopexin-heme complexes[END_REF][START_REF] Madsen | Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region[END_REF]. Indeed, we found that the Hp level in plasma is strongly decreased in SOD1 -/-mice (Fig 2A ). Surprisingly, higher plasma concentrations of Hx were detected in these animals compared with wt littermates. Considering the divergent patterns of Hp and Hx protein abundance in the plasma of SOD1-deficient mice, we analyzed the expression the Hp1 and Hx genes in the liver, the major site of Hp and Hx synthesis [START_REF] Ascenzi | Hemoglobin and heme scavenging[END_REF]. The results shown in Fig 2B indicate that Hp as well as Hx mRNAs are markedly upregulated. We also measured expression of CD163 and CD91 transcripts in the liver, coding for receptors of Hp-Hb and Hx-heme complexes, respectively [START_REF] Kristiansen | Identification of the hemoglobin receptor[END_REF][START_REF] Hvidberg | Identification of the receptor scavenging hemopexin-heme complexes[END_REF]. Levels of CD163 mRNA showed no differences between SOD1 -/-and SOD1 +/+ mice, but the abundance of CD91 mRNA was significantly lower in SOD1-deficient mice (Fig 2C ).

Indices of extravascular hemolysis in SOD1-deficient mice

To investigate the susceptibility of RBC to be removed from the circulation by erythrophagocytosis (EP), as a potential mechanism underlying hemolytic anemia, we evaluated the presence of phosphatydilserine (PS) externalized on the membranes of circulating erythrocytes. Flow-cytometry detection of externalized PS clearly showed a more than 3-fold increase in the percentage of PS-positive SOD1 -/-RBC when compared with wt erythrocytes (Fig 3A). In artificially aged RBC [START_REF] Delaby | A physiological model to study iron recycling in macrophages[END_REF], the proportion of PS translocated to the cell surface was significantly greater than the values found for SOD1 -/-and SOD1 +/+ RBC. We next used an EP assay to examine the correlation between the levels of externalized PS with the heme content in SOD1 -/+ BMDM after phagocytosis of RBC of the two genotypes. As expected, the heme content in BMDM after incubation with SOD1 -/-RBC was significantly greater than after incubation with wt RBC (Fig 3B ) indicating that SOD1 -/-RBC are more readily phagocytosed than SOD1 +/+ RBC.

Increase in hepatic heme oxygenase-1 (HO-1) expression in SOD1 -/-mice To better understand the role of HO-1 during accelerated hemolysis, we analysed its expression in the liver, at both mRNA and protein levels ( were similarly elevated. Interestingly, HO-1 expression in liver was mostly detected in SOD1 - /-Kupffer cells (KC) in accordance to its known high expression in phagocytes. In contrast to wt mice, Hb autofluorescence was strongly present in liver sections from anemic SOD1 -/- mice (Fig 4D). Interestingly, fluorescence analysis of HO-1 (green) with Hb (red) showed partial co-localization in sections of liver from SOD1 -/-mice (Fig 4E).

Hepatic iron status in hemolytic SOD1 -/-mice Massive iron accumulation in tissues is one of the characteristic metabolic features of acute hemolytic anemias of various etiologies [START_REF] Dhaliwal | Hemolytic anemia[END_REF]. We therefore examined whether this iron overload phenotype could be observed in hemolytic SOD1 -/-mice. Staining of non-heme iron in the liver showed iron accumulation in some SOD1 -/-liver macrophages (Fig 5A). On the other hand, no evident sign of global iron loading in hepatocytes was detected. Furthermore, we observed no significant change in non-heme iron content in the liver (Fig 5B). Accordingly, a significant decrease in L-ferritin protein (Fig 5C ) in hepatic cytosolic extracts from SOD1 -/-mice was observed. The protein level of H-ferritin shows no difference between SOD1 +/+ and SOD1 -/-mice.

Hepatic hepcidin (Hepc) mRNA and ferroportin (Fpn) protein levels are respectively decreased and increased in SOD1-deficient mice

We next studied the expression of Fpn, the only known iron exporter in mammalian cells. Western blotting of hepatic membrane extracts (Fig 6A ) and immunofluorescence on histological liver sections (Fig 6B ), showed that Fpn is expressed at low levels in the livers of SOD1 +/+ mice. In contrast, Fpn expression was markedly enhanced in the livers of SOD1 -/- mice (Fig 6A and6B). Microscope analysis of immunofluorescence staining on liver sections (Fig 6B ) identified KC as the principal cell type of Fpn expression. Enlargement of immunofluorescent pictures also indicate the presence of RBC Hb autofluorescence in SOD1 - /-KC strongly positive for Fpn (see enlargement in Fig 6B). At the level of mRNA, we detected a slight but not significant change in Fpn mRNA expression between SOD1 +/+ and SOD1 -/-animals (Fig 6C). Considering the established role of Hepc in the degradation of Fpn molecules [START_REF] De | The molecular mechanism of hepcidin-mediated ferroportin down-regulation[END_REF], we then analyzed Hepc mRNA expression in mice of both genotypes and found that in SOD1 -/-mice, the expression of Hepc mRNA was decreased by about 70% (Fig 6D ).

DISCUSSION

Here, we have investigated the consequences of oxidative stress due to the combined effects of SOD1 deficiency [START_REF] Sentman | Phenotypes of mice lacking extracellular superoxide dismutase and copper-and zinc-containing superoxide dismutase[END_REF][START_REF] Elchuri | CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life[END_REF][START_REF] Iuchi | Elevated oxidative stress in erythrocytes due to an SOD1 deficiency causes and triggers autoantibody production[END_REF] and age-dependent alterations in redox state [START_REF] Sentman | Phenotypes of mice lacking extracellular superoxide dismutase and copper-and zinc-containing superoxide dismutase[END_REF][START_REF] Elchuri | CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life[END_REF] on iron homeostasis in old mice. Considering the well-known susceptibility of RBC to oxidative stress, and the importance of the liver in recycling heme iron, we focused our study on the peripheral erythrocyte-liver axis. Furthermore, analysis in the spleen of SOD1 -/-mice of markers of heme-iron recycling (HO-1, Fpn) and of the macrophage phagosome maturation (lyzosome-associated membrane protein 1, Lamp1) indicate no major changes in heme-iron recycling in this tissue (supplementary data, Fig 1). Our first observation concerning 1-year-old mice lacking Cu,Zn-SOD activity was reduction in the values of the main RBC parameters. A similar level of anemia has been reported in 40week-old SOD1 -/-mice [START_REF] Iuchi | Elevated oxidative stress in erythrocytes due to an SOD1 deficiency causes and triggers autoantibody production[END_REF]. It has been proposed that oxidative stress is the major factor causing SOD1 deficiency anemia to qualify as a hemolytic anemia (HA) [START_REF] Iuchi | Elevated oxidative stress in erythrocytes due to an SOD1 deficiency causes and triggers autoantibody production[END_REF]. Because the mechanisms underlying HA are believed to rely on the destruction or accelerated removal of RBC from the circulation, we looked for evidence of intra-and extravascular hemolysis in old SOD1 -/-mice.

Hemoglobin (Hb) and heme are the most abundant iron compounds released into the plasma from ruptured RBC and they have been implicated in toxic effects [START_REF] Kumar | Free heme toxicity and its detoxification systems in human[END_REF]. Haptoglobin (Hp) and hemopexin (Hx), being scavengers of Hb and heme, respectively, are considered plasma-protective proteins against intravascular hemolysis [START_REF] Ascenzi | Hemoglobin and heme scavenging[END_REF]. The increased hepatic Hp gene expression and reduced plasma Hp protein level we observed in SOD1 -/-mice may reflect the induction of Hp synthesis in the liver, its release into the circulation followed by formation of the Hp-Hb complex, and its clearance by KC via CD163 [START_REF] Kristiansen | Identification of the hemoglobin receptor[END_REF]. Accordingly, the plasma level of Hp is usually reduced in hemeolytic states [START_REF] Ascenzi | Hemoglobin and heme scavenging[END_REF]. With regard to Hx, the increased levels found in the plasma of SOD1 -/-mice could be due to the fact that the amount consumed by moderate hemolysis is outweighed by its enhanced production as a result of inflammation in the liver. Furthermore, plasma Hx level starts to decrease when the Hbbinding capacity of Hp is exceeded [START_REF] Delanghe | Hemopexin: a review of biological aspects and the role in laboratory medicine[END_REF]. Finally, low hepatic expression of CD91 mRNA (suggesting low protein expression) may also explain the high levels of Hx detected in the plasma of SOD1 -/-mice. In support of a role for enhanced extravascular hemolysis, i.e., accelerated erythrophgocytosis (EP), in HA in mice lacking SOD1, we observed the presence of RBC Hb autofluorescence in liver macrophages attesting of the high EP activity of these cells although Hb autofluorescence signal may also derive from internalized Hp-Hb complexes. We also demonstrated marked externalization of phosphatidylserine (PS) on the surface of RBC from SOD1 -/-mice that likely contribute to this observation. Indeed, under physiological conditions the exposure of PS on the RBC membrane is a signal for macrophages to recognize damaged or aged RBC [START_REF] Bratosin | Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages[END_REF]. A role for extravascular hemolysis in the mechanism contributing to HA is further supported ex vivo by the high heme content in BMDM after incubation with RBC from SOD1 -/-mice. This observation clearly indicated the increased susceptibility of SOD1 -/-RBC to be engulfed by phagocytes. In this study, we characterize large changes in hepatic iron metabolism in old SOD1-deficient mice displaying mild but chronic HA. We first analyzed the expression of heme oxygenase-1 (HO-1), an enzyme responsible for heme catabolism in macrophages [START_REF] Maines | The heme oxygenase system: update 2005[END_REF]. We found a substantial increase in HO-1 mRNA and protein expression in the liver of SOD1 -/-mice in comparison to the wt mice. In addition, HO-1 was found to be localized in KC, which exhibited strong Hb fluorescence from ingested RBC. In accordance with our data, increased hepatic HO-1 expression has also been reported in various hemolytic states [START_REF] Immenschuh | Marked hyperbilirubinemia associated with the heme oxygenase-1 gene promoter microsatellite polymorphism in a boy with autoimmune hemolytic anemia[END_REF][START_REF] Tolosano | Enhanced splenomegaly and severe liver inflammation in haptoglobin/hemopexin double-null mice after acute hemolysis[END_REF], and in macrophages in an EP assay [START_REF] Delaby | A physiological model to study iron recycling in macrophages[END_REF]. The role of HO-1 in systemic iron turnover has been confirmed in HO-1 deficiency [START_REF] Poss | Heme oxygenase 1 is required for mammalian iron utilization[END_REF][START_REF] Yachie | Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency[END_REF]. The anemia and hepatic iron overload observed in HO-1-deficient subjects demonstrate the importance of this enzyme in recycling heme iron. On the other hand, it has been reported that potentiated HO-1 expression does not counteract tissue iron loading in acute hemolytic anemia [START_REF] Tolosano | Enhanced splenomegaly and severe liver inflammation in haptoglobin/hemopexin double-null mice after acute hemolysis[END_REF][START_REF] Khan | Hemeoxygenase-mediated iron accumulation in the liver[END_REF]. We found that the moderate HA occurring in SOD1 -/-mice is associated with hepatic deposits of non-heme iron in KC. Therefore, the increased expression of HO-1 in SOD1 -/-liver macrophages likely participates in the iron release from heme into the cytosol and its subsequent storage into ferritin deposits observed in these cells. We observed also a strong upregulation of the iron exporter Fpn in KC that have ingested RBC. The macrophage nature of Fpn positive liver cells in SOD1 -/-mice, has been also confirmed by immunofluorescent co-localization of F4/80 (widely used as a marker for mouse macrophages) with ferroportin (data not shown). Indeed, Fpn is highly expressed in tissue macrophages including KC [START_REF] Abboud | A novel mammalian iron-regulated protein involved in intracellular iron metabolism[END_REF][START_REF] Mckie | A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation[END_REF][START_REF] Donovan | Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter[END_REF]. Hepcidin (Hepc), a peptide secreted by the liver, binds to Fpn and causes its degradation [START_REF] De | The molecular mechanism of hepcidin-mediated ferroportin down-regulation[END_REF]. We found that Hepc levels were reduced in SOD1 -/-mice. It has been proposed that the occurrence of elevated erythropoiesis is sufficient to explain the decreased expression of Hepc by an as yet unidentified erythroid regulator negatively controlling Hepc synthesis [START_REF] Vokurka | Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis[END_REF]. Indeed, enhanced erythropoietic activity as a compensatory reaction in HA in SOD1 -/-has been demonstrated by means of increased reticulocyte counts and splenomegaly [this study , 10]. Interestingly, expansion of the splenic erythroid pool has been reported in erythropoietin-and phenylhydrazine-treated mice [START_REF] Canonne-Hergaux | Characterization of the iron transporter DMT1 (NRAMP2/DCT1) in red blood cells of normal and anemic mk/mk mice[END_REF]. In addition, Fpn expression was shown to be enhanced after EP in macrophages [START_REF] Delaby | A physiological model to study iron recycling in macrophages[END_REF][START_REF] Knutson | Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages[END_REF]. Therefore, we propose that the up-regulation of Fpn detected in KC of SOD1-deficient mice is the consequence of decreased Hepc level and enhanced phagocytosis of SOD1 -/-RBC. Although the coordinated down-and upregulation of Hepc and Fpn, respectively, in the liver is well-documented [START_REF] Delaby | Presence of the exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and down-regulated by hepcidin[END_REF][START_REF] Viatte | Deregulation of proteins involved in iron metabolism in hepcidin-deficient mice[END_REF][START_REF] Peyssonnaux | Regulation of iron homeostasis by the hypoxiainducible transcription factors (HIFs)[END_REF], its relevance for iron flux through this organ has not been clearly considered. In our mouse model of mild HA, the increased levels of hepatic Fpn protein of SOD1 -/-mice may suggest iron release from the liver and in particular from KC. In consequence, serum iron concentration is maintained at normal level despite increased iron demand for erythropoiesis. Consistent with this hypothesis, overexpression of Fpn in tissue culture cells was found to result in increased iron efflux and cellular iron depletion [START_REF] Abboud | A novel mammalian iron-regulated protein involved in intracellular iron metabolism[END_REF][START_REF] Knutson | Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages[END_REF]. Of particular interest, overexpression of Fpn in J774 macrophages was associated with increase in the export of iron after EP [START_REF] Knutson | Iron release from macrophages after erythrophagocytosis is up-regulated by ferroportin 1 overexpression and down-regulated by hepcidin[END_REF]. Interestingly, no iron deposit was found in hepatocytes and L-ferritin, mostly expressed in these cells [START_REF] Doolittle | Isoferritins in rat Kupffer cells, hepatocytes, and extrahepatic macrophages. Biosynthesis in cell suspensions and cultures in response to iron[END_REF], tends to decrease in SOD1 -/-liver. However, several observations indicate that decrease of hepatic L-Ft in SOD1-deficient mice reflects hepatocytic secretion in response to inflammation [START_REF] Tran | Secretion of ferritin by rat hepatoma cells and its regulation by inflammatory cytokines and iron[END_REF] rather than iron depletion in these cells via upregulation of Fpn. Despite clear mRNA expression of the Fpn1 gene in hepatocytes [START_REF] Zhang | Localization of iron metabolism-related mRNAs in rat liver indicate that HFE is expressed predominantly in hepatocytes[END_REF], the protein expression and its subcellular localization in these cells remain poorly documented [START_REF] Viatte | Deregulation of proteins involved in iron metabolism in hepcidin-deficient mice[END_REF]. Furthermore, in mice lacking Hamp gene, an upregulation of Fpn in liver did not prevent iron overload in hepatocytes whereas KC were iron deficient [START_REF] Viatte | Deregulation of proteins involved in iron metabolism in hepcidin-deficient mice[END_REF][START_REF] Nicolas | Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice[END_REF]. Finally, in contrast to KC we did not detect any specific labeling of Fpn in SOD1 -/-hepatocytes.

In summary, we have characterized large changes in iron and heme metabolism triggered in response to modest HA induced by oxidative stress due to SOD1 deficiency and age-related decrease in antioxidant defense. The changes act to reduce the toxicity of Hb and/or heme released from oxidatively damaged RBC through efficient clearance of these iron compounds from circulation. In addition, combined upregulation of HO-1 and Fpn in KC of SOD1 -/-mice suggest that at least a part of heme iron is recycled into the circulation likely to respond to the needs of erythropoiesis. was supported by grant from Polish Ministry of Sciences and Higher Education [N30102431/0668].

FOOTNOTES

The abbreviations used are: BMDM, bone marrow-derived macrophages; EP, erythrophagocytosis; Fpn, ferroportin; Ft, ferritin; heme oxygenase-1; HA, hemolytic anemia; Hepc, hepcidin; KC, Kupffer cells; PS, phosphatidylserine; RBC, red blood cells; SOD1, superoxide dismutase 1;

FIGURE LEGENDS

Table 1 Changes in the hematological parameters of 1-year-old SOD1 -/-mice. Values are expressed as the mean ± SD. Blood cell indices were determined for 10 mice of each genotype. RBC, red blood cells number; Hb, hemoglobin; HCT, hematocrit; MCV, mean cell volume, MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration. * denotes significant differences between SOD1 -/-and SOD1 +/+ mice, P <0.05. . Each column represents the mean ± S.D. of two amplification reactions, performed using a single cDNA sample reversetranscribed from RNA prepared from four mice of each genotype. To confirm amplification specificity, the PCR products from each primer pair were subjected to melting curve analysis and subsequent agarose gel electrophoresis. The levels of Hp and Hx mRNA in SOD1 +/+ mice were assigned as the 100% values. N=4, (P <0.05). (C) Hepatic CD163 and CD91 mRNA levels measured by Real-Time RT-PCR. For description see B.

Figure 3 Indices of increased extravascular hemolysis in SOD1-deficient mice. (A)

Increased externalization of phosphatidylserine (PS) on SOD1 -/-erythrocytes. Exposure of PS was assessed by staining cells with Annexin V-FITC followed by flow cytometry. Artificially-aged, Ca 2+ ionophore A23187-treated RBCs from SOD1 +/+ mice were used as control cells showing high levels of PS exposure on their cell membrane. Stained RBC as a percentage of the total number of erythrocytes was calculated. Results are shown as the mean ± SD for 3 wild-type mice and 4 SOD1 -/-mice. Significant differences in PS exposure are indicated. (B) Increased heme content of bone marrow-derived macrophages (BMDM) after in vitro phagocytosis of erythrocytes. The erythrophagocytosis assay was performed using BMDM from SOD1 -/+ mice and red blood cells (RBC) from 1-year-old SOD1 -/-and SOD1 +/+ mice. Heme content of the formic acid-solubilized BMDM was determined spectrophotometrically at 398 nm. Values represent the means ± SD of 4 erythrophagocytosis assays performed in duplicate on RBC from 4 mice of each genotype. Significant differences in PS exposure are indicated. (P<0.05).

Figure 4 Increased hepatic heme oxygenase-1 (HO-1) expression in SOD1 -/-mice. (A)

Hepatic HO-1 mRNA level. Transcript abundance in livers was measured by real-time RT-PCR using the specific primer pair shown in supplementary data (Table 1). Reactions and data Immunofluorescence staining of Fpn and RBC (hemoglobin autofluorescence) in liver sections from SOD1 -/-and SOD1 +/+ mice (original magnification 63x). Lower panels show enlargement of Fpn staining in Kupffer cells of wild-type (+/+) or SOD1-deficient (-/-) mice. In SOD1 -/-mice Fpn was detected in liver macrophages containing RBC. (C) Expression of ferroportin mRNA. Transcript abundance in livers of SOD1 -/-and SOD1 +/+ mice was measured by real-time RT-PCR using the specific primer pair shown in supplementary data (Table 1). Reactions and data presentation, as described in the legend to Fig 2B . (D) Expression of hepcidin mRNA. Transcript abundance in livers of SOD1 -/-and SOD1 +/+ mice was measured by real-time RT-PCR using the specific primer pair shown in supplementary data Table 1). Reactions and data analysis as described in the legend to Fig 2B, except that six mice of each genotype were used. * Denotes significant differences between hepcidin mRNA levels in the livers of SOD1 -/-and SOD1 +/+ mice (P <0.05). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 1 Changes in the hematological parameters of 1-year-old

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 18 Mar 2009 as manuscript BJ20082137 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082137

  Fig 1A) and transferrin (Fig 1B) may suggest that the anemia in SOD1 -/-animals is not associated with iron deficiency. The raised number of peripheral reticulocytes in SOD1 -/-mice (Fig 1C) as well as the increased spleen index values (Fig 1D) indicate that the anemia in SOD1 -/-mice is regenerative.

  Fig 4). Levels of HO-1 mRNA were increased about 3-fold in SOD1 -/-mice compared with wt littermates (Fig 4A), and HO-1 protein levels assessed by both Western blotting (Fig 4B) and immunofluorescence (Fig 4C)

Biochemical

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Figure 1

 1 Figure 1 Evidence of regenerative and not associated with iron deficiency anemia in SOD1 -/-mice. (A) Increased number of reticulocytes in peripheral blood of SOD1 -/-mice. (B) Increased spleen index in SOD1 -/-mice. Spleen index (SI) was calculated according to the formula SI = weight body weight spleen _ / _ * 100 [45]. (C) Serum iron and (D) serum transferrin levels in SOD1 -/-and SOD1 +/+ mice, N=8-10 for each genotype. * Denotes significant differences between values for SOD1 -/-and SOD1 +/+ mice, (P <0.05).

Figure 2

 2 Figure 2Changes in the expression of haptoglobin (Hp), hemopexin (Hx) and their receptors in SOD1 -/-mice. (A) Plasma levels of Hp and Hx were assessed by Western blotting. For Hp detection, proteins from 1 µl of plasma were separated by SDSpolyacrylamide gel electrophoresis. For detection of Hx, 2 µl of plasma was diluted in 40 µl of loading buffer and then 5 µl was loaded on a SDS-polyacrylamide gel. Plasma from LPStreated SOD1 +/+ mice and from Hx-null mice (kindly provided by Dr. E. Tolosano, University of Turin) were used as positive and negative controls, respectively. Red Ponceau staining of transferred proteins is shown (lower panels) to confirm equivalent loading. (B) Hepatic Hp and Hx mRNA levels. Transcript abundance in livers was measured by Real-Time RT-PCR using specific primer pairs shown in supplementary data (Fig1). Each column represents the mean ± S.D. of two amplification reactions, performed using a single cDNA sample reversetranscribed from RNA prepared from four mice of each genotype. To confirm amplification specificity, the PCR products from each primer pair were subjected to melting curve analysis and subsequent agarose gel electrophoresis. The levels of Hp and Hx mRNA in SOD1 +/+ mice were assigned as the 100% values. N=4, (P <0.05). (C) Hepatic CD163 and CD91 mRNA levels measured by Real-Time RT-PCR. For description see B.

Biochemical

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  presentation, as described in the legend to Fig 1B. (B) HO-1 protein levels as assessed by Western blotting. Membrane proteins from liver (100 µg/lane) were separated by SDSpolyacrylamide gel electrophoresis. An identical blot reacted with actin antibody is shown (lower panel) to confirm equivalent loading. (C) Immunofluorescence staining of HO-1 and (D) autofluorescence of hemoglobin in SOD1 -/-(-/-) and SOD1 +/+ (+/+) livers analyzed by fluorescence microscopy (original magnification 60x). (E) Enlargements of fluorescent staining in SOD1 -/-liver show HO-1 expression in hemoglobin-containing Kupffer cells.

Figure 5

 5 Figure 5 Hepatic iron status in SOD1 -/-mice and wild-type littermates. (A) Histological examination of iron loading in liver. Non-heme iron deposits were detected by staining with Perls Prussian blue (blue stain) exclusively in Kupffer cells of SOD1 -/-mice (arrows). A high magnification of a Kupffer cells presenting clear deposit of iron likely in ferritin is shown (inset in the bottom right panel). (B) Non-heme hepatic iron content was measured as described under Experimental. Values are expressed as the mean ± SD for liver samples obtained from 10 mice of each genotype, (P <0.05). (C) H-and L-ferritin levels in liver cytosolic protein extracts (50 µg/lane) were assessed by Western Blotting. The analyses were performed in quadruplicate using extracts obtained from different SOD1 -/-and SOD1 +/+ mice, and representative results are shown.

Figure 6

 6 Figure 6Correlation between enhanced ferroportin (Fpn) and decreased hepcidin expression in the liver of SOD1 -/-mice. (A) Ferroportin levels in liver membrane protein extracts (100 µg/lane) as assessed by Western blotting. Red Ponceau staining of transferred proteins and an identical blot reacted with actin antibody are shown (lower panels) to confirm equivalent loading. (B) Immunofluorescence staining of Fpn and RBC (hemoglobin autofluorescence) in liver sections from SOD1 -/-and SOD1 +/+ mice (original magnification 63x). Lower panels show enlargement of Fpn staining in Kupffer cells of wild-type (+/+) or SOD1-deficient (-/-) mice. In SOD1 -/-mice Fpn was detected in liver macrophages containing RBC. (C) Expression of ferroportin mRNA. Transcript abundance in livers of SOD1 -/-and SOD1 +/+ mice was measured by real-time RT-PCR using the specific primer pair shown in supplementary data (Table1). Reactions and data presentation, as described in the legend to Fig 2B.(D) Expression of hepcidin mRNA. Transcript abundance in livers of SOD1 -/-and SOD1 +/+ mice was measured by real-time RT-PCR using the specific primer pair shown in supplementary data Table1). Reactions and data analysis as described in the legend to Fig 2B, except that six mice of each genotype were used. * Denotes significant differences between hepcidin mRNA levels in the livers of SOD1 -/-and SOD1 +/+ mice (P <0.05).

  0.5* 9.8 ± 0.9* 32.1 ± 3.5* 58.0 ± 2.5* 17.8 ± 0.3 30.7 ± 0.9Values are expressed as the mean ± SD. Blood cell indices were determined for 10 mice of each genotype. RBC, red blood cells number; Hb, hemoglobin; HCT, hematocrit; MCV, mean cell volume, MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration. * denotes significant differences between Sod1 -/-and Sod1 +/+ mice, P <0.05. Biochemical Journal Immediate Publication. Published on 18 Mar 2009 as manuscript BJ20082137 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082137A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Figure 1

  Figure 4

' 2009 The Authors Journal compilation ' 2009 Portland Press Limited

ACKNOWLEDGMENTS

We would like to thank Drs. C. Bouton and J-C. Drapier (ICSN-CNRS,Gif-sur-Yvette, France) and Dr. S. Imbeaud and L. Marisa (Gif-Orsay Microarray Platform, CNRS, Gif-sur-Yvette, France) for enabling us to compare the iron-related gene expression profiles of aged SOD1 -/-vs SOD1 +/+ mice by DNA microarray technology, which was helpful for designing the experiments described in this manuscript. We thank Olivier Thibaudeau for help in histological analysis. We thank John Gittins for critical reading the manuscript. This work

B.

A. Licenced copy. Copying is not permitted, with prior permission and as allowed by law.


I Jardin

L J Gómez

G M Salido

Dr Juan A Rosado
email: jarosado@unex.es

Dynamic interaction of hTRPC6 with the Orai1/STIM1 complex or hTRPC3 mediates its role in capacitative or non-capacitative Ca 2+ entry pathways

Keywords: hTRPC6, STIM1, Orai1, hTRPC3, calcium entry, thrombin, platelets

TRPC channel subunits have been shown to assemble into homo-or heteromeric channel complexes, including different Ca 2+ -handling proteins, required for the activation of capacitative Ca 2+ entry (CCE) or non-capacitative Ca 2+ entry (NCCE) pathways. Here we present evidence for the dynamic interaction between endogenously expressed hTRPC6 with either both Orai1 and STIM1 or hTRPC3 to participate in CCE or NCCE. Electrotransjection of cells with anti-hTRPC6 antibody, directed towards the C-terminal region, reduces CCE induced by N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), which reduces intraluminal free Ca 2+ concentration. Cell stimulation with thrombin or extensive Ca 2+ store depletion by thapsigargin (TG) + Iono enhanced interaction between hTRPC6 and the CCE proteins Orai1 and STIM1. In contrast, stimulation with the diacylglycerol analogue 1oleoyl-2-acetyl-sn-glycerol (OAG) displaces hTRPC6 from Orai1 and STIM1 and enhances the association between hTRPC6 and hTRPC3. The interaction between hTRPC6 and hTRPC3 was abolished by dimethyl-BAPTA loading, which indicate that this phenomenon is Ca 2+ -dependent. These findings support that hTRPC6 participate both in CCE and NCCE through its interaction with the Orai1/STIM1 complex or hTRPC3, respectively.

INTRODUCTION

The canonical transient receptor potential (TRPC) cation channels are mammalian homologues of the photoreceptor TRP channel in Drosophila melanogaster [START_REF] Montell | The venerable inveterate invertebrate TRP channels[END_REF]. A number of evidences have supported the role of TRPC channels in both capacitative Ca 2+ entry (CCE) and non-capacitative Ca 2+ entry (NCCE) pathways [START_REF] Cheng | Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels[END_REF][START_REF] Liao | Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels[END_REF][START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF][START_REF] Vazquez | Obligatory role of Src kinase in the signaling mechanism for TRPC3 cation channels[END_REF]. The identification of the endoplasmic reticulum (ER) Ca 2+ sensor STIM1 and the plasma membrane (PM) Ca 2+ channel Orai1 has shed new light on the mechanism of activation of CCE. Orai1 forms a highly Ca 2+ selective channel that has been proposed to conduct I CRAC [START_REF] Vig | CRACM1 is a plasma membrane protein essential for store-operated Ca 2+ entry[END_REF]. Orai1 has been reported to participate in the formation of multimeric ion-channel complexes involving hTRPC1 and STIM1 [START_REF] Ong | Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components[END_REF][START_REF] Jardin | Orai1 Mediates the Interaction between STIM1 and hTRPC1 and Regulates the Mode of Activation of hTRPC1forming Ca 2+ Channels[END_REF]. STIM1 is a low affinity Ca 2+ -binding protein mostly located in the ER membrane that contains an EF-hand domain in the NH 2 terminus, located in the lumen of the ER, that has been proposed to function as a Ca 2+ sensor in the ER [START_REF] Roos | STIM1, an essential and conserved component of store-operated Ca 2+ channel function[END_REF][START_REF] Mercer | Large store-operated calcium selective currents due to coexpression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1[END_REF], conferring TRPC1 protein store-operated sensitivity [START_REF] Jardin | Orai1 Mediates the Interaction between STIM1 and hTRPC1 and Regulates the Mode of Activation of hTRPC1forming Ca 2+ Channels[END_REF]. hTRPC6 has been presented as a component of NCCE, as well as CCE, channels. hTRPC6 was initially presented as a channel activated by diacylglycerol (DAG) generated by receptor-induced phosholipase C activation [START_REF] Thebault | Receptor-operated Ca 2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line[END_REF]12]. Furthermore, in human platelets, heteromeric interaction of hTRPC6 with hTRPC1 forms a complex activated by depletion of the intracellular Ca 2+ stores [START_REF] Redondo | Intracellular Ca 2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP 3 receptor and SERCA3 in human platelets[END_REF]. Consistent with this, we have previously reported that hTRPC6 is required for thapsigargin (TG)-mediated Ca 2+ entry in platelets [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF]. In addition, in smooth muscle cells, hTRPC6 channels are activated by mechanical stretch, leading to membrane depolarization and the activation of voltage-dependent Ca 2+ entry [14]. Therefore, TRPC6-forming channels function as integrators of a number of intracellular signals.

There is significant controversy concerning the mode of activation (capacitative or noncapacitative) of particular TRPC channel subunits [START_REF] Parekh | Store-operated calcium channels[END_REF]. These discrepancies might at least be partially attributed to the tetrameric composition of TRPC channels. Thus, different combinations of TRPC subunits might lead to the formation of channels with different biophysical properties. The assembly of a particular combination of TRPC subunits might be performed in situ under an adequate stoichiometric relationship, conditions that might be altered in TRPC-overexpressing cells [START_REF] Parekh | Store-operated calcium channels[END_REF], which supports the importance of experiments in cells endogenously expressing TRPC proteins. In HEK-293 cells expressing TRPC3, TRPC6 and the three Orai isoforms, interaction of Orai proteins with both TRPC proteins has been reported to confer them the ability to respond to store depletion [START_REF] Liao | Orai proteins interact with TRPC channels and confer responsiveness to store depletion[END_REF].

In the present study we have investigated the interaction of endogenously expressed hTRPC6 with components of the CCE and NCCE pathways at resting conditions and upon store depletion either by pharmacological tools or the physiological agonist thrombin or after stimulation with the diacylglycerol analogue OAG. Our results indicate that Ca 2+ store depletion stimulates rapid interaction between hTRPC6 and both, Orai1 and STIM1, while stimulation with the NCCE inducer OAG evokes the displacement of hTRPC6 from the CCE pathway and promotes its interaction with the NCCE Ca 2+ permeable channel, hTRPC3.

MATERIAL AND METHODS

Materials

Fura-2 acetoxymethyl ester (fura-2/AM) and calcein-AM were from Molecular Probes (Leiden, The Netherlands). Apyrase (grade VII), aspirin, thapsigargin (TG), thrombin, phenylmethanesulphonylfluoride (PMSF) and bovine serum albumin (BSA) were from Sigma (Madrid, Spain). Ionomycin (Iono), N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were from Calbiochem (Nottingham, U.K.). Goat anti-hTRPC6 polyclonal antibody, horseradish peroxidaseconjugated goat anti-rabbit IgG antibody and horseradish peroxidase-conjugated donkey anti-goat IgG antibody were from Santa Cruz (Santa Cruz, CA, U.S.A.). Rabbit anti-TRPC1 antibody was from Alomone Labs (Jerusalem, Israel). Rabbit anti-TRPC3 polyclonal antibody was from Abcam (Cambridge, U.K.). Rabbit anti-Orai1 antibody was from ProSci (Poway, CA, U.S.A.). Mouse anti STIM1 antibody was from BD Transduction Laboratories (Frankin Lakes, NJ, U.S.A.). Hyperfilm ECL was from Amersham (Buckinghamshire, U.K.). Protein A-agarose was from Upstate Biotechnology Inc. (Madrid, Spain). Enhanced chemiluminescence detection reagents and rabbit anti-mouse IgG were from Pierce (Cheshire, U. K.). All other reagents were of analytical grade.

Platelet Preparation

Fura-2-loaded platelets were prepared as previously described [START_REF] Redondo | Hydrogen peroxide and peroxynitrite enhance Ca 2+ mobilization and aggregation in platelets from type 2 diabetic patients[END_REF] as approved by Local Ethical Committees and in accordance with the Declaration of Helsinki. Briefly, blood was obtained from healthy drug-free volunteers and mixed with one-sixth volume of acid/citrate dextrose anticoagulant containing (in mM): 85 sodium citrate, 78 citric acid and 111 Dglucose. Platelet-rich plasma (PRP) was then prepared by centrifugation for 5 min at 700 g and aspirin (100 µM) and apyrase (40 µg/mL) were added. Platelet-rich plasma was incubated at 37 ºC with 2 µM fura-2 acetoxymethyl ester for 45 min. Cells were then collected by centrifugation at 350 g for 20 min and resuspended in HEPES-buffered saline (HBS), pH 7.45, containing (in mM): 145 NaCl, 10 HEPES, 10 D-glucose, 5 KCl, 1 MgSO4 and supplemented with 0.1% BSA and 40 µg/mL apyrase.

Cell viability

Cell viability was assessed using calcein and trypan blue. For calcein loading, cells were incubated for 30 min with 5 µM calcein-AM at 37 ºC, centrifuged and the pellet was resuspended in fresh HBS. Fluorescence was recorded from 2 mL aliquots using a Cary Eclipse Spectrophotometer (Varian Ltd, Madrid, Spain). Samples were excited at 494 nm and the resulting fluorescence was measured at 535 nm. The results obtained with calcein were confirmed using the trypan blue exclusion technique.

Measurement of cytosolic free calcium concentration ([Ca 2+ ] c )

Fluorescence was recorded from 2 mL aliquots of magnetically stirred platelet suspension (2 × 10 8 cells/mL) at 37 °C using a fluorescence spectrophotometer with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca 2+ ] c were monitored using the fura-2 340/380 fluorescence ratio and, when TPEN was absent, calibrated according to the method of Grynkiewicz et al. [18]. Ca 2+ entry was estimated using the integral of the rise in [Ca 2+ ] c for 2.5 min after addition of CaCl 2 [19]. Control experiments were performed for all experimental procedures in order to correct the amount of Ca 2+ entry (integrals) by subtraction of the integral of the [Ca 2+ ] c elevation due to leakage of the indicator. Ca 2+ release from the intracellular stores was estimated using the integral of the rise in [Ca 2+ ] c for 3 min after the addition of thrombin.

Reversible Electroporation Procedure

The platelet suspension was transferred to an electroporation chamber containing antibodies at a final concentration of 2 µg/mL, and the antibodies were transjected according to published methods [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF][START_REF] Watson | Platelets : a practical approach[END_REF]. Reversible electropermeabilization was performed at 4 kV/cm at a setting of 25-microfarad capacitance and was achieved by 7 pulses using a Bio-Rad Gene Pulser Xcell Electroporation System (Bio-Rad, Hercules, CA, U.S.A.). Following electroporation, cells were incubated with antibodies for an additional 60 min at 37 ºC then were centrifuged at 350 g for 20 min and resuspended in HBS containing 200 µM CaCl 2 . At the time of experiment 250 µM EGTA was added.

Immunoprecipitation and Western blotting

The immunoprecipitation and Western blotting were performed as described previously [21]. Briefly, 500 µL aliquots of platelet suspension (2 × 10 9 cell/mL) were lysed with an equal volume of RIPA buffer, pH 7.2, containing 316 mM NaCl, 20 mM Tris, 2 mM EGTA, 0.2 % SDS, 2% sodium deoxycholate, 2 % triton X-100, 2 mM Na 3 VO 4 , 2 mM PMSF, 100 µg/mL leupeptin and 10 mM benzamidine. Aliquots of platelet lysates (1 mL) were immunoprecipitated by incubation with 2 µg of either anti-Orai1, anti-TRPC3 or anti-TRPC6 antibody and 25 µL of protein A-agarose overnight at 4 °C on a rocking platform. The immunoprecipitates were resolved by 10 % SDS-PAGE and separated proteins were electrophoretically transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10 % (w/v) BSA in tris-buffered saline with 0.1 % Tween 20 (TBST) to block residual protein binding sites. Immunodetection of STIM1, hTRPC3, hTRPC6 and Orai1 was achieved using the anti-STIM1 antibody diluted 1:250 in TBST for 2h or the anti-hTRPC3, anti-hTRPC6 or anti-Orai1 antibody diluted 1:1000 in TBST for 2h, respectively. The primary antibody was removed and blots were washed six times for 5 min each with TBST. To detect the primary antibody, blots were incubated for 45 min with horseradish peroxidase-conjugated donkey anti-goat IgG antibody, horseradish peroxidaseconjugated rabbit anti-mouse IgG antibody or horseradish peroxidase-conjugated goat antirabbit IgG antibody diluted 1:10000 in TBST and then exposed to enhanced chemiluminiscence reagents for 4 min. Blots were then exposed to photographic films. The density of bands on the film was measured using scanning densitometry. Data were normalized to the amount of protein recovered by the antibody used for the immunoprecipitation.

Statistical Analysis

Analysis of statistical significance was performed using one-way analysis of variance. For comparison between two groups Student's t test was used. P<0.05 was considered to be significant for a difference.

RESULTS hTRPC6 co-immunoprecipitates with STIM1 and Orai1 in human platelets

A role for endogenously expressed hTRPC6 in agonist-induced Ca 2+ entry has been demonstrated in human platelets [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF]12]. We have now investigated the association between hTRPC6 and both, STIM1 and Orai1, by looking for co-immunoprecipitation from platelet lysates. Immunoprecipitation and subsequent SDS-PAGE and Western blotting were conducted using resting platelets and platelets stimulated in the absence of extracellular Ca 2+ (100 µM EGTA was added to the extracellular medium) with the physiological agonist thrombin (1U/mL) or with the inhibitor of the sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA) TG (1 µM) plus a low concentration of Iono (50 nM), to induce extensive depletion of the intracellular stores in platelets [START_REF] Rosado | Two pathways for store-mediated calcium entry differentially dependent on the actin cytoskeleton in human platelets[END_REF]. After treatment with these agents cell viability in our platelet preparations was estimated as 95%. After immunoprecipitation with anti-hTRPC6 antibody, Western blotting with anti-STIM1 antibody revealed a band between 84 and 90 kDa (the predicted molecular weight of STIM1 [START_REF] Manji | STIM1: a novel phosphoprotein located at the cell surface[END_REF]) in samples from resting platelets (Figure 1A, upper panel). Treatment with thrombin increased the association between STIM1 and hTRPC6 in a time-dependent manner, reaching a maximal effect after 30 s of platelet stimulation (Figure 1A, upper panel; n=6). In addition, we have investigated the possible interaction between hTRPC6 and Orai1. After immunoprecipitation with anti-hTRPC6 antibody, Western blotting with anti-Orai1 antibody revealed a band of approximately 45 kDa (the predicted molecular weight of Orai1 [START_REF] Gwack | Biochemical and functional characterization of Orai proteins[END_REF]) in samples from resting platelets (Figure 1B, upper panel). Treatment with thrombin enhanced the coimmunoprecipitation of hTRPC6 with Orai1 in a time-dependent manner (Figure 1B, upper panel; n=6). Similar results were observed when cells were stimulated with TG+Iono, which increased coimmunoprecipitation of hTRPC6 with STIM1 and Orai1 in a time-dependent manner, reaching a maximum after 10 s of stimulation, and then was sustained for at least 60 s (Figure 2A and B, upper panel; n=6). We also conducted converse experiments, immunoprecipitating platelet lysates with anti-STIM1 or anti-Orai1 antibodies and detecting for the presence of hTRPC6, a protein of approximately 100 kDa [START_REF] Zhang | A novel TRPM2 isoform inhibits calcium influx and susceptibility to cell death[END_REF]. Under these conditions, treatment with thrombin or TG+Iono enhanced the association of hTRPC6 with STIM1 in a time-dependent manner, with a 148 ± 24, 195 ± 34 and 179 ± 37% of control for thrombin and 143 ± 18, 155 ± 31 and 126 ± 31% of control for TG+Iono at 10, 30 and 60 s, respectively. Similarly, both agents enhanced the association between hTRPC6 and Orai1 with a 115 ± 6, 150 ± 22 and 180 ± 40% of control for thrombin and 135 ± 17, 158 ± 16 and 131 ± 21% of control for TG+Iono at 10, 30 and 60 s, respectively (Figures 1 and2, C and D upper panels). Western blotting of the same membranes with the antibody used for immunoprecipitation confirmed similar protein content in all lanes (Figures 1 and2, lower panels). No specific proteins were detected when immunoprecipitation was performed using a non-specific rabbit IgG control. Only the heavy chain of the rabbit IgG used for immunoprecipitation was detected when a horseradish peroxidase-conjugated anti-rabbit IgG was used as secondary antibody in lanes 3 to 5 (Supplementary Figure 1).

hTRPC6 is involved in TPEN-mediated Ca 2+ entry

TPEN may be a useful tool to investigate the mechanism of CCE due to its ability to reduce intraluminal free Ca 2+ concentration. The use of TPEN avoids potential confounders, such as changes in cytosolic Ca 2+ and second messenger levels, secondary to stimulation with agonists, as well as the use of pharmacological inhibitors of SERCA, which has been suggested to play a role in CCE [START_REF] Redondo | SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets[END_REF]. In a Ca 2+ -free medium, treatment of fura-2-loaded human platelets with thrombin (1U/mL) in stirred cuvettes at 37 °C evoked a transient elevation in [Ca 2+ ] c due to Ca 2+ release from internal stores. Subsequent addition of 300 µM Ca 2+ to the external medium induced a sustained increase in [Ca 2+ ] c (Figure 3A upper graph; n=5). TPEN is a membrane-permeant multivalent cation chelator, that shows moderate affinity for Ca 2+ (Kd for Ca 2+ ~130 µM), which has been used to rapidly and reversibly chelate Ca 2+ within the intracellular stores [START_REF] Hofer | Capacitative Ca2+ entry is closely linked to the filling state of internal Ca 2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+][END_REF]. In the absence of extracellular Ca 2+ , the addition of 500 µM TPEN to human platelets did not modify [Ca 2+ ] c ; subsequent addition of 300 µM Ca 2+ to the external medium induced a sustained elevation in [Ca 2+ ] c indicative of SOCE [START_REF] Iwasaki | 2-Aminoethoxydiphenyl borate (2-APB) inhibits capacitative calcium entry independently of the function of inositol 1,4,5-trisphosphate receptors[END_REF], due to in situ chelation of Ca 2+ in the intracellular stores (Figure 3A upper graph; n=5). The elevation in Fura-2 fluorescence ratio observed after the addition of Ca 2+ to the medium in the presence of TPEN was clearly greater than that observed in the absence of TPEN (Figure 3A lower graph; n=5). In the presence of TPEN, thrombin-evoked Ca 2+ release and entry were significantly reduced (Figure 3A upper graph), a phenomenon that is not likely attributed to a decrease in cell viability (95% of cells were viable in our platelet preparations treated with TPEN, at least during the performance of the experiments). A c c e p t e d M a n u s c r i p t antibody, directed towards an intracellular C-terminal sequence, following a previously described procedure [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF]. Platelet viability after reversible electroporation was 90%; therefore experimental cells and controls were treated in the same way, except for the antibody electrotransjected (anti-hTRPC3 or hTRPC6 antibodies in experimental cells and a nonspecific rabbit IgG in controls). We have recently reported that platelet incubation with the anti-hTRPC6 antibody reduced Ca 2+ and Mn 2+ entry induced by thrombin, TG or 2,5-di-(tert-butyl)-1,4-hydroquinone, thus suggesting a role for hTRPC6 in CCE [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF]. Here we show for the first time that electrotransjection with 1 µg/mL hTRPC6 significantly reduced TPENevoked CCE by 33 ± 6% compared to controls (cells electrotransjected with 1 µg/mL goat IgG; Figure 3B upper graph; P<0.05; n=5). We have further explored the role of hTRPC3 in CCE induced by TPEN by electrotransjection of cells with anti-hTRPC3 antibody directed towards the intracellular NH 2 -terminal sequence. As shown in Figure 3B upper graph, TPEN-evoked CCE was not modified in cells electrotransjected with 1 µg/mL anti-hTRPC3 antibody compared to platelets electrotransjected with 1 µg/mL rabbit IgG. Finally, we found that electrotransjection of the anti-hTRPC6 antibody in combination with anti-hTRPC3 antibody reduced CCE by TPEN to a similar extent as the anti-hTRPC6 antibody alone (Figure 3B upper graph). As mentioned in material and methods, the amount of Ca 2+ entry was corrected by subtraction of the elevation in [Ca 2+ ] c observed after the addition of Ca 2+ in cells treated with vehicle instead of thrombin and/or TPEN (Figure 3A and B lower graphs).

Ca 2+ -dependent association of hTRPC6 and hTRPC3

We have further investigated the interaction between hTRPC6 and hTRPC3 upon stimulation with thrombin or TG + Iono. Immunoprecipitation with anti-hTRPC6 antibody followed by Western blotting with anti-hTRPC3 antibody revealed a band of approximately 100 kDa (the predicted molecular weight of hTRPC3 [START_REF] Brenner | TrpC3 regulates hypertrophy-associated gene expression without affecting myocyte beating or cell size[END_REF]), which indicates association of both hTRPC proteins in resting platelets (Figure 4 upper panels). In the absence of extracellular Ca 2+ , platelet stimulation with thrombin or extensive Ca 2+ store depletion, induced by TG + Iono, enhanced co-immunoprecipitation between hTRPC6 and hTRPC3 (Figure 4A and B upper panels; P<0.05). This effect was abolished by platelet loading with the intracellular Ca 2+ chelator dimethyl BAPTA, which prevents thrombin-, as well as TG + Iono-induced Ca 2+ elevations in [Ca 2+ ] c (Figure 4C-F). Similar results were observed when converse experiments were conducted immunoprecipitating platelet lysates with anti-hTRPC3 antibody and detecting for the presence of hTRPC6. Treatment with thrombin or TG+Iono enhanced the association of hTRPC6 with hTRPC3 by 142 ± 8 and 151 ± 11% of control, respectively (Figure 4A andB, lower panels). This event was prevented by BAPTA loading (Figure 4E and F, lower panels).

Interestingly, we have found that OAG, a DAG analogue that activates NCCE, attenuates the association between hTRPC6 and both, Orai1 and STIM1, as detected by immunoprecipitation of platelet lysates with anti-hTRPC6 antibody followed by Western blotting with anti-Orai1 or anti-STIM1 antibodies (Figure 5A and B, upper panels; P<0.05) as well as by converse experiments (Figure 5A and B, lower panels; P<0.05). In the absence of extracellular Ca 2+ , OAG was unable to induce association between hTRPC6 and hTRPC3 (Figure 5C). In contrast, in the presence of 1 mM extracellular Ca 2+ OAG significantly enhanced association between both hTRPC proteins (Figure 5D; P<0.05). Western blotting of the same membranes with the anti-hTRPC6 antibody confirmed comparable amounts of protein in all lanes (Figures 4 and5).

As shown in Figure 5E upper graph, treatment of platelets in a Ca 2+ -free medium with 100 µM OAG did not increase [Ca 2+ ] c ; subsequent addition of Ca 2+ to the external medium resulted in a rise in [Ca 2+ ] c , greater than that induced in cells treated with vehicle alone (Figure 5E lower graph), indicative of NCCE. Electrotransjection with 1 µg/mL hTRPC3 or hTRPC6 significantly reduced OAG-evoked NCCE by 40 ± 9 and 56 ± 2%, respectively (Figures 5G andH) compared to controls (cells electrotransjected with 1 µg/mL rabbit IgG; Figure 5F(α-mouse IgG); P<0.05; n=5). Altogether, these findings suggest that the association of hTRPC6 and hTRPC3 induced by OAG is mediated by a Ca 2+ -dependent mechanism, as previously described for the association between both proteins stimulated by thrombin or TG + Iono.

Treatment with TG + ionomycin is unable to induce association of hTRPC3 with hTRPC1, STIM1 or Orai1

We have further investigated the possible interaction between hTRPC3 and hTRPC1, STIM1 and Orai1, which form a dynamic complex involved in CCE [START_REF] Jardin | Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1forming Ca 2+ channels[END_REF], upon stimulation with TG + Iono. Immunoprecipitation with anti-hTRPC3 antibody followed by Western blotting with anti-hTRPC1 antibody revealed a faint band of approximately 100 kDa (the predicted molecular weight of hTRPC1 [31, 32]), which indicates certain association of both hTRPC proteins in resting platelets (Figure 6A upper panel; n=5). After platelet stimulation with TG + Iono the amount of hTRPC1 immunoprecipitated with the anti-hTRPC3 antibody was not significantly different from than observed in resting cells (Figure 6A upper panel). No protein with the predicted molecular weight of STIM1 or Orai1 were detected in hTRPC3 immunoprecipitates either in resting or in TG+Iono-stimulated cells (Figure 6B andC, upper panels; n=5). Similar results were observed when converse experiments were conducted immunoprecipitating platelet lysates with anti-hTRPC1, anti-STIM1 or anti-Orai1 antibody and detecting for the presence of hTRPC3 (Figure 6 lower panels).

DISCUSSION

Canonical TRP proteins have been shown to participate both in capacitative and noncapacitative Ca 2+ influx [START_REF] Parekh | Store-operated calcium channels[END_REF]. hTRPC6 has long been proposed as a candidate to mediate NCCE in different cell types [START_REF] Thebault | Receptor-operated Ca 2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line[END_REF][START_REF] Boulay | Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein[END_REF][START_REF] Trebak | The TRPC3/6/7 subfamily of cation channels[END_REF], including human platelets [12]. In these cells, we have recently suggested that hTRPC6 might also be involved in the conduction of CCE on the base of the inhibitory effect of electrotransjection of the anti-hTRPC6 antibody on TGinduced Ca 2+ entry [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF]. CCE is a mechanism for Ca 2+ influx regulated by the Ca 2+ content of the intracellular stores rather than by the Ca 2+ signals generated by agonists or pharmacological tools like TG [START_REF] Putney | A model for receptor-regulated calcium entry[END_REF]. Here we have further observed the involvement of hTRPC6 in CCE by using TPEN, an intracellular Ca 2+ chelator that reduces free Ca 2+ concentration in the intracellular stores ([Ca 2+ ] s ) without changes in [Ca 2+ ] c secondary to stimulation with agonists or TG, which confirms previous results [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF]. We have found that hTRPC3, which has been shown to conduct CCE in a number of cell types [36,[START_REF] Vazquez | Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent storeoperated cation channels in DT40 avian B lymphocytes[END_REF] but NCCE in others [START_REF] Zitt | Expression of TRPC3 in Chinese hamster ovary cells results in calciumactivated cation currents not related to store depletion[END_REF][START_REF] Zhu | Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca 2+ entry[END_REF], is not required for Ca 2+ entry induced by TPEN, as demonstrated by electrotransjection of the anti-hTRPC3 antibody, which further suggest that treatment with TPEN induces CCE in human platelets. The specificity of electrotransjection with antibodies has been explored by testing the effect of incubation with an antibody directed to a protein not related to hTRPC6 or hTRPC3 proteins or any other platelet protein. We have used goat or rabbit IgG since these are the natures of the anti-hTRPC6 and anti-hTRPC3 antibodies, respectively. Thrombin-evoked Ca 2+ release and entry were significantly reduced in the presence of TPEN. TPEN has been shown to cause a rapid decrease in [Ca 2+ ] s [START_REF] Caroppo | A reassessment of the effects of luminal [Ca 2+ ] on inositol 1,4,5trisphosphate-induced Ca 2+ release from internal stores[END_REF], where Ca 2+ concentration is elevated (~0.2-1 mM [START_REF] Verkhratsky | Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons[END_REF]), which might explain the significant reduction of the extent of Ca 2+ release induced thrombin. The inhibitory effect of TPEN on Ca 2+ entry mediated by thrombin might be partially attributed to its chelating effects and to the known activation of Ca 2+ extrusion through the Na + /Ca 2+ exchanger [START_REF] Shmist | N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine improves myocardial protection against ischemia by modulation of intracellular Ca 2+ homeostasis[END_REF].

CCE has been shown to require a dynamic interplay between the ER Ca 2+ sensor STIM1 and the PM channels Orai1 and hTRPC1 [START_REF] Ong | Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components[END_REF]. In human platelets we have recently reported that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential to confer hTRPC1 properties of capacitative Ca 2+ entry channel [START_REF] Jardin | Orai1 Mediates the Interaction between STIM1 and hTRPC1 and Regulates the Mode of Activation of hTRPC1forming Ca 2+ Channels[END_REF]. Here we present for the first time evidence for the interaction between endogenously expressed hTRPC6 and both Orai1 and STIM1 in platelets. The association between these proteins was detected in resting platelets and enhanced by the physiological agonist thrombin or extensive store depletion using TG + Iono. The interaction between hTRPC6 and both Orai1 and STIM1 was found to be independent on extracellular Ca 2+ and might mediate the involvement of hTRPC6 in CCE in these cells.

On the basis that hTRPC6 and hTRPC3 have been shown to be involved in NCCE and that both channels have been reported to be associated to the same platelet membrane regions [43], we have explored the interaction between both proteins upon stimulation with thrombin or TG + Iono and OAG, a DAG analogue that activates NCCE [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF]. Platelet treatment with thrombin or TG + Iono enhances interaction between hTRPC6 and hTRPC3 only when rises in [Ca 2+ ] c are allowed, a response that was abolished by cell loading with dimethyl-BAPTA. Since capacitative Ca 2+ entry was first proposed as a mechanism for Ca 2+ influx regulated by the Ca 2+ content of the intracellular stores rather than by the Ca 2+ signals generated by agonists [START_REF] Putney | A model for receptor-regulated calcium entry[END_REF], our findings suggest that this interaction is non-capacitative in nature due to its dependence on rises in [Ca 2+ ] c . Similarly, stimulation with OAG increases the association of hTRPC6 with hTRPC3 when Ca 2+ entry is allowed, suggesting that this interaction is Ca 2+dependent. In addition, this finding suggests that a different mechanism mediates the initial Ca 2+ entry step activated by OAG. Interestingly, treatment with OAG decreased the interaction of hTRPC6 with Orai1 and STIM1, which indicates that activation of NCCE pathways results in displacement of hTRPC6 from the CCE protein complex to the NCCE mechanism. To further support in role of hTRPC3 in NCCE we have found that store depletion with TG + Iono did not induce association between hTRPC3 with hTRPC1, STIM1 or Orai1.

Our findings suggest that hTRPC6 interacts with the CCE channel protein Orai1 and the ER Ca 2+ sensor STIM1 upon stimulation with the physiological agonist thrombin or extensive Ca 2+ store depletion. hTRPC6 is required for CCE induced either by TG [START_REF] Jardin | Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets[END_REF] or TPEN, which induces a decrease in [Ca 2+ ] s but avoiding rises in [Ca 2+ ] c . The NCCE activator OAG displaces hTRPC6 from Orai1 and STIM1 and induces its association with hTRPC3. We propose that depletion of [Ca 2+ ] s induces the formation of a macromolecular complex involving plasma membrane Ca 2+ -permeable channels like Orai1, hTRPC6 and hTRPC1 [START_REF] Jardin | Orai1 Mediates the Interaction between STIM1 and hTRPC1 and Regulates the Mode of Activation of hTRPC1forming Ca 2+ Channels[END_REF]. In contrast, activation of NCCE results in displacement of hTRPC6 from the CCE pathway and favors Ca 2+ -dependent association between hTRPC6 and hTRPC3 (a schematic diagram of the proposed model is depicted in Figure 7). These data support that hTRPC6 participates in CCE and NCCE pathways in human platelets by association to different Ca 2+ -handling proteins. A-D, Human platelets (2 × 10 9 cell/mL) were suspended in a Ca 2+ -free medium (100 µM EGTA added) and then stimulated for various periods of time (10-60 s) with thrombin (1 U/mL) and lysed. Whole cell lysates were immunoprecipitated (IP) with anti-hTRPC6 (A and B), anti-STIM1 (C) or anti-Orai1 antibody (D). Immunoprecipitates were subjected to 10 % SDS-PAGE and subsequent Western blotting with a specific anti-STIM1 (A), anti-Orai1 (B) or anti-hTRPC6 antibody (C and D). Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control. The panel shows results from one experiment representative of 5 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Histograms represent the quantification of hTRPC6-STIM1 association (A) or hTRPC6-Orai1 association (B) in resting (control) and thrombin-treated cells. Results are presented as arbitrary optical density units and expressed as mean ± S.E.M. * P<0.05 vs. control. Human platelets (2 × 10 9 cell/mL) were suspended in a Ca 2+ -free medium (100 µM EGTA added) and then stimulated for various periods of time (10-60 s) with TG (1µM) + Iono (50 nM) and lysed. Whole cell lysates were immunoprecipitated (IP) with anti-hTRPC6 (A and B), anti-STIM1 (C) or anti-Orai1 antibody (D) and immunoprecipitates were subjected to 10 % SDS-PAGE and subsequent Western blotting with a specific anti-STIM1 (A) anti-Orai1 (B) or anti-hTRPC6 antibody (C and D). Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control. The panel shows results from one experiment representative of 5 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Histograms represent the quantification of hTRPC6-STIM1 association (A) or hTRPC6-Orai1 association (B) in resting (control) and thrombin-treated cells. Results are presented as arbitrary optical density units and expressed as mean ± S.E.M. * P<0.05 vs. control. (A) Fura-2-loaded human platelets were suspended in a Ca 2+ -free medium (100 µM EGTA was added) and treated with TPEN (500 µM; upper graph) or the vehicle (DMSO, lower graph), as control. Platelets were stimulated with 1 U/mL thrombin (upper graph) or the vehicle (HBS, lower graph) 1 min later followed by the addition of CaCl 2 (final concentration 300 µM) 3 min later to initiate Ca 2+ entry. (B) Human platelets were electropermeabilized in a Gene Pulser as described in "material and methods" and then were incubated in the presence of 1 µg/mL anti-hTRPC6 antibody, anti-hTRPC3 antibody, both or 1 µg/mL rabbit or goat IgG (Control upper graph and lower graph) for 60 min as indicated. Cells were then stimulated in a Ca 2+ -free medium (250 µM EGTA was required since electroporated cells were suspended in medium containing 200 µM CaCl 2 , see material and Control (A and B) and dimethyl BAPTA-loaded (E and F) human platelets (2 × 10 9 cell/mL) were suspended in a Ca 2+ -free medium (100 µM EGTA added) and then stimulated for 30 s with thrombin (1U/mL; A and E) or TG (1µM) + Iono (50 nM; B and F) and lysed. Whole cell lysates were immunoprecipitated (IP) with anti-hTRPC6 antibody (A, B, E and F upper panels) or anti-hTRPC3 antibody (A, B, E and F lower panels) and immunoprecipitates were subjected to 10 % SDS-PAGE and subsequent Western blotting with a specific anti-hTRPC6 (upper panels) or anti-hTRPC3 (lower panels) antibody. Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control. The panels show results from one experiment representative of 5 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Histograms represent the quantification of hTRPC6-hTRPC3 association in resting (control) and stimulated cells. Results are presented as arbitrary optical density units and expressed as mean ± S.E.M. * P<0.05 vs. control. (C and D) Human platelets were incubated at 37 °C for 30 min in the presence or absence of 10 µM dimethyl BAPTA, as indicated. At the time of experiment 100 µM EGTA was added. Cells were then stimulated with 1 U/mL thrombin (C) or 1 µM TG + 50 nM Iono (D). Elevations in [Ca 2+ ] c were monitored using the 340/380 nm ratio as described in the materials and methods section. Traces shown are representative of 5 independent experiments. A-D, Human platelets (2 × 10 9 cell/mL) were suspended in a Ca 2+ -free medium (100 µM EGTA added; A-C) or in a medium containing 1 mM CaCl 2 (D) and then stimulated for 30 s with OAG (100 µM) and lysed. Whole cell lysates were immunoprecipitated (IP) with anti-hTRPC6 antibody and immunoprecipitates were subjected to 10 % SDS-PAGE and subsequent Western blotting with a specific anti-hTRPC6 (A-D, upper panels), anti-Orai1 (A, lower panel), anti-STIM1 (B, lower panel) or anti-hTRPC3 antibody (C and D, lower panels). Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control. The panel shows results from one experiment representative of 5 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Histograms represent the quantification of the association of hTRPC6 with Orai1 (A), STIM1 (B) and hTRPC3 (C and D) in resting (control) and OAGstimulated cells. Results are presented as arbitrary optical density units and expressed as mean ± S.E.M. * P<0.05 vs. control. (E-H) Human platelets were electropermeabilized in a Gene Pulser as described in "material and methods" and then were incubated in the absence (E) or presence of 1 µg/mL anti-hTRPC6 antibody (G), anti-hTRPC3 antibody (H), or rabbit anti-mouse IgG (F) for 60 min as indicated. Platelets were suspended in a Ca 2+ -free medium (250 µM EGTA was added) and stimulated with OAG (100 µM; E upper graph, F, G and H) or the vehicle (DMSO; E lower graph), as control, followed by the addition of CaCl 2 (final concentration 1 mM) 3 min later to initiate Ca 2+ entry. Human platelets (2 × 10 9 cell/mL) were suspended in a Ca 2+ -free medium (100 µM EGTA added) and then stimulated for 30 s with TG (1µM) + Iono (50 nM) and lysed. Whole cell lysates were immunoprecipitated (IP) with anti-hTRPC3 (upper panels), anti-hTRPC1 (A, lower panel), anti-STIM1 (B, lower panel) or anti-Orai1 antibody (C, lower panel) and immunoprecipitates were subjected to 10 % SDS-PAGE and subsequent Western blotting with a specific anti-hTRPC1 (A, upper panel), anti-STIM1 (B, upper panel), anti-Orai1 (C, upper panel) or anti-hTRPC3 antibody (lower panels). Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control. The panel shows results from one experiment representative of 5 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Histograms (A) represent the quantification of the association of hTRPC1 with hTRPC3 in resting (control) and TG+Iono-stimulated cells. Results are presented as arbitrary optical density units and expressed as mean ± S.E.M. Activation of phospholipase C (PLC), via G protein-coupled receptors, results in the synthesis of IP 3 , which, in turn, activates IP 3 receptors (IP 3 R) in the endoplasmic reticulum (ER). Depletion of the ER leads to the association of hTRPC6 to Orai1 and STIM1 (presented as the ER Ca 2+ sensor) to participate in store-operated or capacitative Ca 2+ entry (CCE). In contrast, activation of a non-capacitative Ca 2+ entry (NCCE) pathway (i.e. via generation of DAG) displaces hTRPC6 from Orai1 and STIM1 and induces its association with hTRPC3.

  To investigate the involvement of hTRPC6 in TPEN-mediated CCE we have induced functional knock down of hTRPC6 by electrotransjection of cells with anti-hTRPC6 5 Biochemical Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20082179 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082179

Biochemical

  Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20082179 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082179 A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. Time-course of thrombin-induced hTRPC6 co-immunoprecipitation with STIM1 and Orai1 in human platelets.

Figure 2 .

 2 Figure 2. Time-course of TG + ionomycin-induced hTRPC6 co-immunoprecipitation with STIM1 and Orai1 in human platelets.

Figure 3 .

 3 Figure 3. TPEN-mediated Ca 2+ entry is attenuated by electroporation of the anti-hTRPC6 antibody.

13 Biochemical

 13 Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20082179 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082179 A c c e p t e d M a n u s c r i p t methods) with 500 µM TPEN (upper graph) or the vehicle (DMSO, lower graph) and 3 min later CaCl 2 (1 mM) was added to the medium to initiate Ca 2+ entry. The traces are representative of five independent experiments.

Figure 4 .

 4 Figure 4. Thrombin and extensive Ca 2+ store depletion induced by TG + ionomycin induce Ca 2+ -dependent co-immunoprecipitation of hTRPC6 with hTRPC3.

Figure 5 .

 5 Figure 5. OAG enhances co-immunoprecipitation of hTRPC6 with hTRPC3 in the presence of extracellular Ca 2+ .

Figure 6 .

 6 Figure 6. Treatment with TG + ionomycin is unable to induce co-immunoprecipitation of hTRPC3 with hTRPC1, STIM1 or Orai1.

Figure 7 .

 7 Figure 7. Speculative model of the involvement of hTRPC6 in capacitative and noncapacitative Ca 2+ entry in platelets.

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ACKNOWLEDGEMENTS

This work was supported by MEC grant BFU2007-60104. I.J. was supported by a MEC fellowship (BFU2007-60104).

FOOTNOTES

The abbreviations used are: [Ca 2+ ] c , cytosolic free calcium concentration; [Ca 2+ ] s , free Ca 2+ concentration in the intracellular stores; BSA, bovine serum albumin; CCE, capacitative Ca 2+ entry; DAG, diacylglycerol; ER, endoplasmic reticulum; HBS, HEPES-buffered saline; hTRPC6, human canonical TRP6; Iono, ionomycin; IP 3 R, inositol 1,4,5-trisphosphate receptor; NCCE, non-capacitative Ca 2+ entry; OAG, 1-oleoyl-2-acetyl-sn-glycerol; PBS, phosphate-buffered saline; PLC, phospholipase C; PM, plasma membrane; PMSF, phenyl methyl sulphonyl fluoride; STIM1, stromal interaction molecule 1; TBST, tris-buffered saline with 0.1 % Tween 20; TG, thapsigargin; TPEN, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene diamine; TRP, transient receptor potential.