Stephan Reitinger

Gerhard Thomas Laschober

Christine Fehrer

Brigitte Greiderer

Günter Lepperdinger
email: guenter.lepperdinger@oeaw.ac.at

MOUSE TESTICULAR HYALURONIDASE-LIKE PROTEINS, SPAM1 AND HYAL5 BUT NOT HYALP1 DEGRADE HYALURONAN

Keywords: hyaluronan, hyaluronidase, fertilization, spermatogenesis, extracellular matrix Hyaluronan, HA, extracellular matrix, ECM, glycosylphosphatidylinositol, GPI, glycosaminoglycan, GAG, phospholipase C, PLC

Besides SPAM1 (formerly named PH-20), further hyaluronidase-like proteins, HYAL5 and HYALP1 are also expressed in murine testicular tissue. As they share a high degree of sequence similarity with known hyaluronidases, all three polypeptides could potentially exhibit hyaluronidase activity, a function that is beneficial for sperm in order to penetrate the hyaluronan-rich cumulus, which surrounds the oocyte. Recently, it was reported that SPAM1-deficient mice are fertile and sperm derived from mutant mice still exhibits hyaluronidase activity (Baba et al., (2002) J. Biol. Chem. 277, 30310-30314).

We have now recombinantly expressed mouse SPAM1, HYAL5 and HYALP1 in Xenopus laevis oocytes and determined their respective expression pattern in testis. Transcripts of all three genes are expressed in seminiferous tubules in regions, where maturing spermatogenic cells reside. SPAM1 and HYAL5 but not HYALP1 proteins exhibit hyaluronidase activity at neutral pH. The two active hyaluronidases are both bound to the cell surface via a glycosylphosphatidylinositol anchor. Furthermore, structural characteristics are discussed that are necessary for hyaluronidases in order to exhibit hyaluronan cleavage.

Introduction

Hyaluronan (HA), a polymer consisting of repeating ß-1,4linked disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid, is a common glycosaminoglycan present in the extracellular matrix (ECM) of connective tissues [START_REF] Laurent | The structure and function of hyaluronan: An overview[END_REF]. HA is implicated in many physiological processes, including cell migration, proliferation, and differentiation. Hyaluronidases, enzymes that hydrolyse HA to oligosaccharides, have been identified in mammalian, insect, and bacterial species [START_REF] Kreil | Hyaluronidases -a group of neglected enzymes[END_REF][START_REF] Stern | Devising a pathway for hyaluronan catabolism: are we there yet?[END_REF].

HA accumulates between cumulus cells (4) together with inter-α trypsin inhibitor [START_REF] Chen | Covalent linkage between proteins of the inter-alpha-inhibitor family and hyaluronic acid is mediated by a factor produced by granulosa cells[END_REF] as well as a variety of other matrix proteins [START_REF] Salustri | Hyaluronan and proteoglycans in ovarian follicles[END_REF][START_REF] Camaioni | Proteoglycans and proteins in the extracellular matrix of mouse cumulus cell-oocyte complexes[END_REF][START_REF] Richards | Ovulation: new factors that prepare the oocyte for fertilization[END_REF]. The jelly-like shell protects the oocyte and facilitates its extrusion at ovulation [START_REF] Richards | Delivery of the oocyte from the follicle to the oviduct: a time of vulnerability[END_REF]. Furthermore, the swollen cumulus is more easily captured by the fimbria of the oviduct and thus supports oocyte transportation to the site of fertilization [START_REF] Talbot | Oocyte pickup by the mammalian oviduct[END_REF]. The tight matrix however is difficult to perforate by mechanical means. Sperm hyaluronidase activity positively correlates with fertilization rate, because sperm that possesses high motility and fertilization efficiency penetrates highly viscous solutions of HA at a higher rate [START_REF] Aitken | Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence[END_REF][START_REF] Mortimer | A simplified approach to spermcervical mucus interaction testing using a hyaluronate migration test[END_REF]. Therefore, the motility and sperm hyaluronidase activity are thought to facilitate path finding through the extracellular matrix of cumulus cells, since no proteases have yet been implicated in this process [START_REF] Primakoff | Penetration, adhesion, and fusion in mammalian sperm-egg interaction[END_REF]. A widely conserved mammalian sperm surface protein, PH-20 [START_REF] Myles | Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization[END_REF], also called sperm adhesion molecule 1 (SPAM1) [START_REF] Jones | Expression analysis, genomic structure, and mapping to 7q31 of the human sperm adhesion molecule gene SPAM1[END_REF][START_REF] Zheng | The murine Spam1 gene: RNA expression pattern and lower steady-state levels associated with the Rb(6.16) translocation[END_REF] is well known for its hyaluronidase activity [START_REF] Gmachl | Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm[END_REF][START_REF] Gmachl | The human sperm protein PH-20 has hyaluronidase activity[END_REF]. This glycosylphosphatidylinositol-anchored (GPI) sperm hyaluronidase has multiple roles in fertilization [START_REF] Cherr | The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling[END_REF]. SPAM1 and the soluble form of HYAL2, sHYAL2 exhibit activity at both neutral and acidic pH [START_REF] Vigdorovich | Expression and characterization of a soluble, active form of the jaagsiekte sheep retrovirus receptor, Hyal2[END_REF], whereas for other hyaluronidases, enzymatic activity appears to be restricted to either acidic pH, e.g. HYAL1 (21) or neutral pH as known for Xenopus XKH1 [START_REF] Reitinger | Xenopus kidney hyaluronidase-1 (XKH1), a novel type of membrane-bound hyaluronidase solely degrades hyaluronan at neutral pH(1)[END_REF].

Unexpectedly, mice carrying a null mutation in the Spam1 gene are fertile [START_REF] Baba | Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg[END_REF]. In vitro fertilization assays showed that mouse sperm, lacking SPAM1, possesses a reduced ability to disperse cumulus cells from the cumulus mass, resulting in delayed fertilization solely at the early stages after insemination. In this particular context, sperm extracts derived from mutant mice contain at least one Copyright 2006 Biochemical Society further hyaluronidase, HYAL5, which is active in solutions of both neutral pH and extracellular ionic composition [START_REF] Kim | Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass[END_REF].

In the mouse genome, there are seven hyaluronidase-like sequences, six of them clustered in groups of three at two chromosomal sites, on chromosomes 9 F1-F2 (Hyal1, Hyal2 and Hyal3) and 6 A2 (Hyal4, Spam1/PH-20 and Hyalp1) [START_REF] Baba | Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg[END_REF][START_REF] Csoka | The six hyaluronidase-like genes in the human and mouse genomes[END_REF][START_REF] Zhang | Sperm dysfunction in the Rb(6.16)-and Rb(6.15)-bearing mice revisited: involvement of Hyalp1 and Hyal5[END_REF]. The orthologous human genes are localized on syntenic regions, on chromosomes 3p21.3 and 7q31.3. HYALP1 is an expressed pseudogene in humans [START_REF] Csoka | Expression analysis of six paralogous human hyaluronidase genes clustered on chromosomes 3p21 and 7q31[END_REF]. In mouse, Hyalp1 shows more characteristics of a gene that codes for a transcript that can be translated into a polypeptide chain similar to SPAM1. The seventh mouse hyaluronidase-like gene, tentatively called Hyal5, is localized in close proximity to the cluster at chromosomal region 6 A2. So far, no further hyaluronidase-like gene has been discovered in the human genome at this position. Both genes, Hyalp1

and Hyal5 encode open reading frames for a polypeptide highly similar to biochemically characterized hyaluronidases [START_REF] Zhang | Sperm dysfunction in the Rb(6.16)-and Rb(6.15)-bearing mice revisited: involvement of Hyalp1 and Hyal5[END_REF]. Moreover, these three genes are strongly expressed in testis. So far, none of these murine gene products has been recombinantly expressed and assayed for enzymatic activity. Hence, HYAL5, HYALP1 or both could actually be functionally involved for fertilization in mouse. We therefore expressed these proteins in frog oocytes and studied their enzymatic properties with respect to HA degradation.

Biochemical Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598

Copyright 2006 Biochemical Society

Material and Methods

Frog surgery, oocytes preparation, cDNA construction, cRNA injection -A small lobe of ovary was surgically removed from an anaesthetized adult Xenopus laevis female. The ovarian tissue was rinsed in O-R2 buffer (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mM Na 2 HPO 4 , 5 mM HEPES, pH 7.8) and individual oocytes were defolliculated manually. The coding regions of Spam1, Hyal5, and Hyalp1 were cloned into pT7TS (gift from P.A. Krieg, Univ. of Arizona, Tucson, AZ, USA), suitable for generating in vitro-transcribed mRNA. Hyal5 cDNA (clone ID IMAGp998B198849.1) was purchased from the German Resource Center Berlin (RZPD). The protein coding region of Spam1 as well as Hyalp1 was amplified by PCR using cDNA derived from mouse testis as a template, and for Spam1 primers 5'-GGTGATCAATGGGAGAGTTGAGATTTAAGCACCTC-3' and 5'-CCGCTAGCC TAAGGAGTACTGACTAGTGTCTTCCG-3', while for Hyalp1, primers 5'-TCGAGATATCATGTTTATCCAGTGGGTGACACAG-3' and 5'-GGACTAGTTTAGAAAATGCTTGAAATCAAAGA-3' were used. About 50 ng of capped cRNA was injected with the aid of a Nanoliter Injector (World Precision Instruments) into oocytes that were then cultured in O-R2 containing 100 U/mL penicillin and 100 µg/mL streptomycin at 16°C for up to 2 days. GPI-linked proteins were removed from the plasma membranes using 50 mU of Bacillus cereus phospholipase C (PLC) (Molecular Probes) in 30 µL of O-R2 buffer, pH 7.0 containing 4 mM CaCl 2 , 50 µg/mL BSA per oocyte at ambient temperature for 1 hour.

Expression analysis -For expression analysis, RNA was extracted as described previously [START_REF] Chomczynski | Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[END_REF]. Mice were dissected. Specimens were carefully rinsed in PBS and snap frozen in liquid nitrogen. Cells or pieces of tissue were homogenized in 4. Copyright 2006 Biochemical Society volume of 2-propanol. The RNA pellet was rinsed with 80% ethanol, dried and dissolved in 6 M urea, 100 mM HEPES pH 7.5. As a final purification step the RNA was precipitated by the addition of 50 (v/v)% 9 M LiCl. 5 µg of total RNA derived from various tissues and organs were separated by 1.2 % agarose gel electrophoresis and subsequently transferred to Zetaprobe nylon membrane (BIORAD). After UVcrosslinking, blots were hybridized with 33 P-labelled cDNA probes. RT-PCR assays were carried out using specific primer pairs for α-actin, Hyal5 and Hyalp1 on template cDNAs derived from a range of C57BL/6J mouse tissues, and from a murine Sertoli cell line, TM4 [START_REF] Mather | Establishment and characterization of two distinct mouse testicular epithelial cell lines[END_REF]. PCR was carried out in volumes of 25 µL, containing 1 µM primer and 2.5 units Pfu DNA polymerase in reaction buffer with MgSO 4 (as recommended by the manufacturer, Fermentas) for 30 and 40 cycles at 95°C for 1 minute, 58°C for 1 minute and 72°C for 1 minute. Subsequently, the products were analyzed on a 2% agarose gel.

Quantitative RT-PCR assays were performed with the aid of a LightCycler instrument and LightCycler 3.5 software (Roche). The reactions were carried out in a volume of 15 µL with the LC-FastStart DNA Master SYBR Green I Kit (Roche), 2 µM primer and 3 mM MgCl 2. After the activation of the enzyme at 95°C for 7 minutes, 45 amplification cycles were performed at 95°C for 15 seconds, 58°C for 8 seconds and 72°C for 12 seconds. The following oligonucleotide primers were used in order to amplify cytoplasmic α-actin: fwd 5'-GGCTGTATTCCCCTCCATCG-3', rev 5'-CCAGTTGGTAACAATGCCATGT-3'; Hyal5: fwd 5'-AAAGACCCTCGAAATCCAC-3', rev 5'-CCACATACCATTCCAGAGG-3'; and Hyalp1: fwd 5'-TGTCACTTTGTGGAGATCGGA-3', rev 5'-GCCAACGTGAGAGTAACATTCA-3'. These primer pairs yielded amplification products of 154 base pairs (bp) for α-actin, 144 bp for Hyal5 and 222 bp for Hyalp1. The relative abundance of Hyal5 and Hyalp1 transcripts was calculated relative to α-actin, and the values were normalized to the expression level in testis.

In situ hybridization was performed as described previously [START_REF] Tontsch | Automated in situ hybridization In[END_REF]. Briefly, testicular tissues from C57BL/6J mice were fixed in phosphate buffered saline containing 4% para-formaldehyde. The tissue was embedded in Paraplast Plus (Sigma), sectioned to a thickness of 7 µm and mounted on poly-L-lysine coated microscope slides. After treatment with proteinaseK and prehybridization, sections were incubated Copyright 2006 Biochemical Society at 50°C with digoxigenin-labelled RNA probes in hybridization buffer containing 50 (v/v)% formamide, 1xSSC, 1x Denhardt's solution, 5 mM EDTA, 50 µg/mL yeast RNA, 0.2% Tween 20, 0.5% CHAPS, 10 µg/mL heparin. Thereafter, sections were washed with hybridization buffer and the probes were detected using anti-digoxigenin alkaline phosphatase Fab conjugate and BM Purple as a substrate (Roche).

Hyaluronidase activity measurement -Enzyme activity was measured as described previously [START_REF] Müllegger | Hapten-labeled hyaluronan, a substrate to monitor hyaluronidase activity by enhanced chemiluminescence-assisted detection on filter blots[END_REF].

Briefly, enzymes were mixed with 5-aminofluorescein-labelled HA and incubated at different pH values (100 mM sodium phosphate buffer containing 50 µg/mL BSA and 0.1% Triton X-100 first adjusted with citric acid to pH 3.5 and then titrated to the appropriate pH with sodium hydroxide). The reaction mixture was separated by 1% agarose gel electrophoresis. The gel was subsequently blotted onto a positively charged nylon membrane (Biodyne PLUS, 0.45 µm, Pall Life Sciences) and the hapten was detected after incubation with anti-fluorescein alkaline phosphatase Fab conjugate (Roche) by means of enhanced chemiluminescence technology (Amersham Biosciences).

Molecular modelling -Tertiary structural models for the hyaluronidase domains of SPAM1, HYAL5 and HYALP1 were generated applying homology modelling of SWISS-MODEL (32) using its alignment interface together with mouse hyaluronidase-like protein sequences of SPAM1 (accession number AAP49832), HYAL5 (accession number BAC55071) and HYALP1 (accession number BAB30316) prealigned with the aid of ClustalW and the X-ray structure of bee hyaluronidase (PDB accession code: 1fcv).

Images were generated with the aid of DeepView/Swiss-PdbViewer version 3.7 [START_REF] Guex | SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling[END_REF].

Biochemical Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598

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RESULTS

Structural Features -SPAM1, HYAL5 and HYALP1 were found to share conserved elements with hyaluronidase from honeybee. Besides the first highly conserved N-terminal domain, all mammalian hyaluronidase-like proteins have a second domain at the C-terminal part of the amino acid sequence. For SPAM1 type hyaluronidases, processing and GPI anchor linkage is needed at the carboxy terminus in order to bind the enzyme to the outer surface of the plasma membrane [START_REF] Phelps | Restricted lateral diffusion of PH-20, a PI-anchored sperm membrane protein[END_REF]. Bioinformatic prediction of whether one of the three different mouse proteins would contain a consensus pattern for GPI linkage, yielded inconclusive results [START_REF] Kronegg | Detection/prediction of GPI cleavage site (GPI-anchor) in a protein[END_REF][START_REF] Eisenhaber | Prediction of potential GPI-modification sites in proprotein sequences[END_REF]. Therefore, it did not become clear from these theoretical considerations, which one of the three hyaluronidase-like genes would potentially code for a HAdegrading enzyme, and thus is functionally involved in fertilization in mouse.

Expression Pattern -Using Northern blots, Hyal5 as well as Hyalp1 were found to be expressed in mouse testis (Fig. 1A). Like Spam1, transcripts of the other two genes are barely detectable within other tissues (Fig. 1B). Applying quantitative reverse-transcription PCR techniques, trace amounts of both Hyal5 and Hyalp1 could be detected in epididymis. This may well be due to sperm present in the highly convoluted epididymal duct. In TM4 Sertoli cell line [START_REF] Mather | Establishment and characterization of two distinct mouse testicular epithelial cell lines[END_REF][START_REF] Gore-Langton | The absence of specific interactions of Sertoli-cell-secreted proteins with antibodies directed against H-Y antigen[END_REF], only Hyal5 transcripts, yet no Hyalp1 transcripts could be detected. A small number of Hyalp1 transcripts as compared to the amount found in testis was also detected in liver, bone marrow, brain, fat, skeletal muscle, lung and spleen as well as in epididymis (Fig. 1B,C). As shown previously, SPAM1 and HYAL5 proteins appear to be present on acrosome intact sperm [START_REF] Baba | Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg[END_REF]. Furthermore, HYALP1 has been found not only in testis and caudal sperm but also in spermfree epididymis [START_REF] Kim | Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass[END_REF].

The precise distribution of Spam1, Hyal5 and Hyalp1 transcripts in testicular tissue, was determined by in situ hybridization (Fig. 2). All three hyaluronidase-like transcripts show an equivalent expression pattern of a ring-shaped area within seminiferous tubules (Fig. 2B-D We therefore deduced from this expression pattern that transcripts of mouse hyaluronidase-like genes are accumulating in spermatogenic cells, most probably in pachytene and/or leptotene spermatocytes (stage I, II, V, and/or VI). No expression was found near or at the basal lamina, where type A and B spermatogonia, preleptotene spermatocytes of stage III and IV reside. Due to the expression pattern, we conclude that all three polypeptides, SPAM1, HYAL5 and/or HYALP1 protein are present in spermatids and could thus also bound to mature sperm.

Enzymatic activity -Since mouse HYAL5, HYALP1 and SPAM1 have not been recombinantly expressed to examine hyaluronidase activity, we translated in vitro transcribed cRNA of all three candidates in intact Xenopus laevis oocytes. Cell lysates were tested for hyaluronidase activity using a sensitive assay [START_REF] Müllegger | Hapten-labeled hyaluronan, a substrate to monitor hyaluronidase activity by enhanced chemiluminescence-assisted detection on filter blots[END_REF].

For HYAL5 and SPAM1, hyaluronidase activity was found in a broad pH range at both acid and neutral conditions. Residual activity could still be measured at pH 7.5 (Fig. 3). In contrast to other hyaluronidases such as HYAL2, which primarily generates intermediate length reaction products of around 20 kD in size, both HYAL5 and SPAM1 were found to degrade HA to short fragments. No hydrolysis was detectable after reaction of HYAL5 and SPAM1 expressing ooyctes with glycosaminoglycans (GAG) such as chondroitine sulfate A, B, C or heparan sulfate as substrates (data not shown). For recombinant HYALP1, no hydrolyzing activity with respect to HA was detectable though Hyalp1 and Hyal5 cRNA were equally well translated in vitro in the presence of rabbit reticulocyte lysate (Fig. 4). When cell-free expression systems together with microsomal preparations were used for the translation of cRNAs that encode wellcharacterized hyaluronidases, in no case were enzymatically active protein products obtained. Hence, we extensively tested HYALP1 expressing oocytes or corresponding culture supernatants, in order to detect the presence of trace amounts of HA-degrading activity. As positive controls, besides SPAM1 and

Biochemical Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598

Copyright 2006 Biochemical Society HYAL5, human PH-20, HYAL1, honeybee hyaluronidase and Xenopus XKH1 [START_REF] Reitinger | Xenopus kidney hyaluronidase-1 (XKH1), a novel type of membrane-bound hyaluronidase solely degrades hyaluronan at neutral pH(1)[END_REF] as well as XHyal2 [START_REF] Müllegger | Degradation of hyaluronan by a Hyal2-type hyaluronidase affects pattern formation of vitelline vessels during embryogenesis of Xenopus laevis[END_REF] were enzymatically active, when expressed in Xenopus oocytes. Yet no HA hydrolyzing activity was detectable after Hyalp1 cRNA injection neither in cells nor in the culture supernatant. Furthermore, chondroitine sulfate A and C as well as heparan sulfate were not depolymerized by HYALP1 expressing oocytes (data not shown).

Next, frog oocytes expressing HYALP1, HYAL5 and SPAM1 were incubated with PLC in order to test whether the enzymes had been linked via GPI anchors to the cell surface. In the cases of HYAL5 and SPAM1, enzyme activity was found in the supernatant of PLC-treated oocytes (Fig. 5). HYAL2, a hyaluronidase that is normally bound to the plasma membrane via a GPI anchor and inactive at neutral pH [START_REF] Rai | Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation[END_REF], was however found to exhibit week enzymatic activity also at neutral pH, when recombinantly expressed in its soluble form, sHYAL2 [START_REF] Vigdorovich | Expression and characterization of a soluble, active form of the jaagsiekte sheep retrovirus receptor, Hyal2[END_REF]. In the case of HYALP1 expressing oocytes, no HA degrading activity was detectable in the supernatant. Notably, the rat homologue of SPAM1, 2B1 glycoprotein (PH20) is also not rendered free from the cell surface by PLC-treatment [START_REF] Seaton | Rat sperm 2B1 glycoprotein (PH20) contains a Cterminal sequence motif for attachment of a glycosyl phosphatidylinositol anchor. Effects of endoproteolytic cleavage on hyaluronidase activity[END_REF]. We therefore have to admit that HYALP1 could be linked to the membrane and only exerts enzymatic activity after being released by a yet unknown process. In conclusion, HYAL5 and SPAM1, after entering the secretory pathway, are sorted and eventually bound through a GPI-anchor to the plasma membrane as active hyaluronidases.

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DISCUSSION

In testicular extracts, hyaluronidase activity has been recognized by biochemical means previously [START_REF] Chain | Identity of hyaluronidase and spreading factor[END_REF]. Molecular cloning of the hyaluronidase present in bee venom [START_REF] Gmachl | Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm[END_REF] revealed strong similarity to a previously known protein that is localized on the posterior head of human sperm, first characterized by means of a monoclonal antibody, and was therefore called PH-20 [START_REF] Myles | Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization[END_REF]. PH-20 from man exhibits strong and robust hyaluronidase activity at neutral pH [START_REF] Gmachl | The human sperm protein PH-20 has hyaluronidase activity[END_REF][START_REF] Lin | A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg[END_REF][START_REF] Lin | Molecular cloning of the human and monkey sperm surface protein PH-20[END_REF]. As recently described, point mutations not only in Spam1, but also in other hyaluronidase-like genes such as Hyal5 and HyalP1 correlate with sperm dysfunction [START_REF] Zhang | Sperm dysfunction in the Rb(6.16)-and Rb(6.15)-bearing mice revisited: involvement of Hyalp1 and Hyal5[END_REF]. As pointed out above, no further gene that potentially would encode hyaluronidase-like proteins others than HYAL1-4 and PH-20/SPAM1 has been discovered in the human genome to date.

Besides mouse, Hyal5 is also present in rat (accession number BC091219). This suggests that further gene duplication has occurred in rodents [START_REF] Zhang | Sperm dysfunction in the Rb(6.16)-and Rb(6.15)-bearing mice revisited: involvement of Hyalp1 and Hyal5[END_REF]. Yet, mature sperm of SPAM1-deficient mice still exhibits hyaluronidase activity. It could be used to fertilize eggs [START_REF] Baba | Mouse sperm lacking cell surface hyaluronidase PH-20 can pass through the layer of cumulus cells and fertilize the egg[END_REF] and is able to disperse cumulus cells from the cumulus mass [START_REF] Kim | Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass[END_REF]. In light of our results, we assume that this is due to functionally active HYAL5 present on the surface of Spam1 -/-sperm. We furthermore conclude, after having shown that SPAM1 as well as HYAL5 are functionally active hyaluronidases, that both enzymes participate in sperm fertilization of the egg.

The biological role for HYALP1 remains to be defined. SPAM1, HYAL5 and HYALP1 (39-64% identical; 64-81% similar) share also identical residues and a high degree of similarity with other well characterized hyaluronidases such as human PH-20 or bee hyaluronidase (supplementary Fig. 1). The most remarkable structural feature of hyaluronidase is a large groove (44) (Fig. 6). The amino acid residues of the groove that were found to come in close proximity to the substrate, are arranged in wellconserved sequence blocks, and, we thus consider them functionally relevant for proper positioning of the sugar polymer. In particular, we propose that four more amino acid moieties (indicated by x in Fig. 6A) that are located in close proximity to the active center, control the positioning of the substrate at the active Copyright 2006 Biochemical Society center. Two amino acid residues were found conserved in all three proteins (yellow-shaded x in Fig. 6A).

The other two are different in HYALP1 (labelled green and blue in Fig. 6A). Molecular modelling revealed a close relationship between the geometries of the active centers of bee hyaluronidase, SPAM1 and HYAL5. However, major spatial differences were evident in the structure of HYALP1, in particular amino acid moieties that appear to be important for the proper positioning of the substrate with regard to the acidic amino acid moieties within the active center Fig. 6B). The active sites of these enzymes consist of aspartate and glutamate interspaced by one amino acid residue [START_REF] Arming | In vitro mutagenesis of PH-20 hyaluronidase from human sperm[END_REF][START_REF] Markovic-Housley | Structural evidence for substrate assisted catalytic mechnism of bee venom hyaluronidase, a major allergen of bee venom In[END_REF], Notably, HYAL5 and SPAM1, yet also HYALP1 show this particular feature. Since both active center residues are present in HYALP1, we assume that the protein, though hydrolysis of HA was undetectable, might exhibit some yet unknown enzymatic activities. For the substrate binding groove of HYALP1 showed a high resemblance to the structures of bee hyaluronidase and the structural models of other known hyaluronidases, binding of HA or other GAG could indeed be possible. Human PH-20 exhibits many more functions besides HA degradation [START_REF] Cherr | The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling[END_REF]. Whether HYALP1 is functionally involved in metabolism of GAG or plays some role during sperm maturation will be investigated in future experiments. (B) On the basis of the X-ray structure of bee hyaluronidase (PDB accession code: 1fcv), 3D-structural models for SPAM1, HYAL5 and HYALP1 were generated (upper row). Residues that were marked with ○, •, ■ and▲and marked as such x in A, were found to be located opposite and in close proximity to the active center (ac) within the tapering substrate binding cleft. These appear to directly interact with the substrate. The 3D-models were slabbed along the line drawn in the frontal view of the bee hyaluronidase structure. Surfaces were drawn in light grey, interior parts of the molecules in dark color. Side projections of the active center reveal variant geometries of the active centers of active hyaluronidases, (bee hyaluronidase, HYAL5 and SPAM1) and inactive HYALP1.

Biochemical

  2 M guanidinium thiocyanate, 100 mM HEPES pH 7.5. The resulting solution was extracted with phenol:chloroform:isoamyl alcohol (25:50:1) and the aqueous phase supplemented with 10 (v/v)% of 2 M sodium acetate, pH 4.4, and 20 (v/v)% water and extracted with phenol:chloroform (3:1). The RNA within the aqueous phase was precipitated by addition of an equal Biochemical Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598

  ). Some of the sectioned tubules show weak to no signals for Spam1, Hyal5 and Hyalp1 expression. For controls, hybridizations with sense probes Biochemical Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598 Copyright 2006 Biochemical Society were performed in parallel. Low background and no specific signals were observed (Fig. 2B-D, inlaid micrographs). In sub-human species, a particular cellular association consisting of spermatogenic cells of a distinct stage occupies a relatively long segment along the tubule. High expression of Hyal5 or Hyalp1 transcripts in Sertoli cells appears unlikely since only minor amounts were detected in cultured TM4 cells.

Biochemical

  Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598

  Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598 Copyright 2006 Biochemical Society

Fig. 1 .

 1 Fig. 1. Expression analysis. Hyal5 and Hyalp1 mRNA expression was determined in various tissues using

Fig. 2 .

 2 Fig. 2. Transcript localization in testicular tissue. (A) Histological section of murine testis stained with

Fig. 3 .Copyright 2006 Biochemical Society 20 Fig. 4 .

 3204 Fig. 3. Hyaluronidase activity of HYAL5 and HYALP1 expressing Xenopus laevis oocytes. Oocytes

Fig. 5 .

 5 Fig. 5. Subcellular localization of HYAL5 and HYALP1. Hyal5, HyalP1 and Spam1 cRNA, respectively,

Fig. 6 .

 6 Fig. 6. Structural models: (A) Mouse hyaluronidase-like protein sequences, SPAM1 (accession number

Biochemical Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598

Biochemical Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598

Acknowledgements

Stephan Reitinger is a DOC fellow of the Austrian Academy of Sciences. Günter Lepperdinger is an APART fellow of the Austrian Academy of Sciences and further supported by the Jubilee Fund of the Austrian National Bank as well as by the Austrian Science Fund, project S9309-B09. We are grateful to Günther Kreil for fruitful discussions and careful reading of the manuscript.

Biochemical

Journal Immediate Publication. Published on 22 Aug 2006 as manuscript BJ20060598 Copyright 2006 Biochemical Society


Rafal Radoslaw Starzynski

Ana Sofia Gonçalves

Françoise Muzeau

Zofia Tyrolczyk

Ewa Smuda

Jean-Claude Drapier

Carole Beaumont

Pawel Lipinski
email: p.lipinski@ighz.pl

STAT5 proteins are involved in

Keywords: EMSA, electrophoretic mobility-shift assay, GAS, gamma interferon activated sequence, IFN-γ -interferon gamma, IRP1, iron regulatory protein 1, LIP, labile iron pool, MRE, metal responsive element, MTF1, metal transcription factor 1, NO, nitric oxide, NOS2, nitric oxide synthase 2, ONPG, o-nitrophenyl-β-D-galactopyranoside, Sp1, iron metabolism, IRP1, nitric oxide, promoter, STAT, transcription

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INTRODUCTION

In mammals, cellular iron homeostasis is largely coordinated at the posttranscriptional level through the action of two cytoplasmic iron regulatory proteins (IRP1 and IRP2). They function by binding to RNA stem-loop structures, known as iron-responsive elements (IREs), located in the 5'-or 3'-untranslated regions, respectively, of specific mRNAs that code for ferritin and transferrin receptor. The activity of IRPs is modulated by changes in intracellular iron concentrations, becoming activated as translational repressors and/or molecules conferring mRNA stability by low iron and inactivated by high levels of iron in the labile iron pool (LIP) [START_REF] Hentze | Balancing acts: molecular control of mammalian iron metabolism[END_REF]. The inactive IRP1 has a secondary function as cytosolic aconitase when coordinating a [4Fe-4S] cluster, which masks the IRE-binding site. It has long been held that IRP1 is a major posttranscriptional regulator that controls iron metabolism in response to fluctuations in intracellular iron. However, recent studies on KO IRP1 [START_REF] Meyron-Holtz | Genetic ablations of iron regulatory proteins 1 and 2 reveal why iron regulatory protein 2 dominates iron homeostasis[END_REF] and KO IRP2 mice [START_REF] Cooperman | Microcytic anemia, erythropoietic protoporphyria, and neurodegeneration in mice with targeted deletion of iron-regulatory protein 2[END_REF][START_REF] Galy | Generation of conditional alleles of the murine Iron Regulatory Protein (IRP)-1 and -2 genes[END_REF] strongly suggest that IRP2, rather than IRP1, is responsible for maintenance of the constitutive body iron balance. The new concept of IRP1 as a molecular target responding preferentially to stimuli other than iron-related challenges is now under debate [START_REF] Bouton | Iron regulatory proteins as NO signal transducers[END_REF][START_REF] Meyron-Holtz | Mammalian tissue oxygen levels modulate iron-regulatory protein activities in vivo[END_REF]. Indeed, the two activities of IRP1 have been shown to be modulated by nitric oxide (NO) [START_REF] Drapier | Biosynthesis of nitric oxide activates iron regulatory factor in macrophages[END_REF][START_REF] Weiss | Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway[END_REF],

oxidative stress [START_REF] Martins | Oxidative stress induces activation of a cytosolic protein responsible for control of iron uptake[END_REF][START_REF] Pantopoulos | Rapid responses to oxidative stress mediated by iron regulatory protein[END_REF], phosphorylation [START_REF] Schalinske | The iron-sulfur cluster of iron regulatory protein 1 modulates the accessibility of RNA binding and phosphorylation sites[END_REF], and hypoxia/reoxygenation [START_REF] Hanson | Regulation of iron regulatory protein 1 during hypoxia and hypoxia/reoxygenation[END_REF].

NO and/or NO-derived species appear to be chemical messengers able to induce a broad spectrum of posttranslational modifications of the IRP1 molecule. First, NO may act directly on the [4Fe-4S] cluster of IRP1, inducing its gradual disassembly and removal, favoring the IRE-binding conformation of the protein [START_REF] Drapier | Redox modulation of iron regulatory proteins by nitric oxide and peroxynitrite[END_REF][START_REF] Soum | Peroxynitrite and nitric oxide differently target the iron-sulfur cluster and amino acid residues of human iron regulatory protein 1[END_REF]. Second, nitration of IRP1, reported in vitro following its exposure to peroxynitrite [START_REF] Soum | Peroxynitrite and nitric oxide differently target the iron-sulfur cluster and amino acid residues of human iron regulatory protein 1[END_REF] and in NO-and superoxide (O 2 .-)-producing macrophages [START_REF] Gonzalez | Endogenous nitration of iron regulatory protein-1 (IRP-1) in nitric oxide-producing murine macrophages: further insight into the mechanism of nitration in vivo and its impact on IRP-1 functions[END_REF] has been shown to prevent IRP1 activation. Lastly, NO is also responsible for the down-regulation of IRP1 expression at both mRNA and protein levels [START_REF] Oliveira | Down-regulation of iron regulatory protein 1 gene expression by nitric oxide[END_REF].

Needless to say that modulation of IRP1 function is important element but not the sole contribution in a complex network of interactions between NO and intracellular iron [reviewed in [START_REF] Watts | Effects of nitrogen monoxide and carbon monoxide on molecular and cellular iron metabolism: mirror-image effector molecules that target iron[END_REF][START_REF] Lipiński | Interplay between ferritin metabolism, reactive oxygen species and nitric oxide[END_REF]. Besides IRP1, IRP2 has been also shown to be regulated by NO [START_REF] Recalcati | Nitric oxide-mediated induction of ferritin synthesis in J774 macrophages by inflammatory cytokines: role of selective iron regulatory protein-2 downregulation[END_REF][START_REF] Wang | Nitric oxide inhibits the degradation of IRP2[END_REF].

It is also important to recall that one of the most apparent hallmarks of NO action originally described by Hibbs et al. [START_REF] Wang | Nitric oxide inhibits the degradation of IRP2[END_REF] is depletion of intracellular iron. Recently, the mechanism of NO-induced iron loss involving glutathione and multidrug resistance-associated protein 1 has been proposed [START_REF] Watts | Nitrogen monoxide (NO)mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1[END_REF]. Another common feature of cells exposed to NO is the formation of ironnitrosyl complexes with multiple heme and non-heme iron proteins inhibiting key enzymes in DNA synthesis and mitochondrial respiration [reviewed in [START_REF] Henry | EPR characterization of molecular targets for NO in mammalian cells and organelles[END_REF][START_REF] Vanin | Dinitrosyl iron complexes and S-nitrosothiols are two possible forms for stabilization and transport of nitric oxide in biological systems[END_REF]. Finally, NO has been reported to be a strong inducer of heme oxygenase expression at both transcriptional [START_REF] Durante | Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells[END_REF] and posttranscriptional level [START_REF] Bouton | Nitric oxide-inducible expression of heme oxygenase-1 in human cells. Translation-independent stabilization of the mRNA and evidence for direct action of nitric oxide[END_REF].

In this study, we focused on NO-mediated down regulation of IRP1, an effect that may be of considerable biological importance, especially under conditions of sustained NO synthesis, because it could reduce the amount of the protein available for posttranslational modification.

The biological mechanisms underlying the NO-induced decrease in IRP1 expression are unknown, although it has been suggested that NO can modify transcriptional activity of the IRP1 gene [START_REF] Oliveira | Down-regulation of iron regulatory protein 1 gene expression by nitric oxide[END_REF]. Indeed, accumulating evidence suggests that NO affects gene expression at the transcriptional level [START_REF] Bogdan | Nitric oxide and the regulation of gene expression[END_REF] by modulating the activities of various transcription factors (TFs) such as NFĸB [START_REF] Shin | Nitric oxide attenuates vascular smooth muscle cell activation by interferon-gamma. The role of constitutive NF-kappa B activity[END_REF], AP-1 [START_REF] Pilz | Nitric oxide and cGMP analogs activate transcription from AP-1-responsive promoters in mammalian cells[END_REF], Sp1 [START_REF] Sellak | Sp1 transcription factor as a molecular target for nitric oxide--and cyclic nucleotide--mediated suppression of cGMP-dependent protein kinase-Ialpha expression in vascular smooth muscle cells[END_REF], HIF-1 [START_REF] Metzen | Nitric oxide impairs normoxic degradation of HIF-1alpha by inhibition of prolyl hydroxylases[END_REF], and STAT (signal transducer and activator of transcription) [START_REF] Bingisser | Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway[END_REF]. Here, we sought to locate putative binding sites for TFs regulated by NO and/or changes in intercellular iron level. Through examination of the nucleotide sequence of the mouse IRP1 promoter (GenBank accession no. AJ427344), we identified the following conserved motifs: one Sp1 sequence, one metal responsive element (MRE) and one binding site for STAT family proteins (GAS sequence, interferon-γ-activated sequence). Functional analysis of IRP1 promoter region and analysis of these putative TF binding sites by electrophoretic mobility shift assay (EMSA) using nuclear extracts from NOproducing RAW 264.7 macrophages indicate that the GAS sequence is an important regulatory element responsible for down-regulation of IRP1 expression by NO. Transfections and reporter gene assays. 24 h before transfection, HuH7 cells were plated in six-well culture plates (NUNC, Denmark) at 50-70% confluence. The cells were transiently transfected with 1 µg of plasmid DNA using Lipofectamine reagent according to the manufacturer's recommendations. 6 h after transfection, cells were incubated with 250 µM DETA/NO for 48 h. After incubation the cells were harvested, total cellular extracts were prepared, and luciferase activity was assessed using the Luciferase Assay System kit according to the manufacturer's instructions (Promega). As an internal control to assess transfection efficiency, cells were co-transfected with a reporter vector (pSV β-gal)

EXPERIMENTAL

containing the β-galactosidase gene driven by the SV40 virus promoter (Promega). Luciferase activities were normalized relative to co-expressed β-galactosidase activity values measured in a standard assay in which ONPG hydrolysis was quantified by absorbance at 420 nm using a Hitachi U-2000 spectrophotometer. As negative and positive controls for transient transfection experiments, the pGL2 basic vector without any promoter sequence and a pGL3 control vector containing the SV40 promoter were used, respectively.

Electrophoretic Mobility Shift Assay (EMSA).

Nuclear protein extracts were prepared from cells as previously described [START_REF] Andrews | A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells[END_REF]. Three double-stranded DNA fragments, created by annealing oligonucleotides, were used as probes: 5`-CCAGGCTCCGCCCCTCGGGG-3`, 5`-CCCCGAGGGGCGGAGCCTGG-3`; 5`-GAAGGGAGGGCGCACCCGGACT-3`, 5`-AGTCCGGGTGCGCCCTCCCTTC-3` and 5`-AAGGCATTTCTAGGAAAATAAGAT-3`, 5`-ATCTTATTTTCCTAGAAATGCCTT-3`. These sequences were identical to the following regions of the mouse IRP1 promoter: -70 to -50 bp containing an Sp1 binding site (underlined), -141 to -119 bp containing an MRE sequence (underlined), and -1363 to -1339 bp containing a GAS sequence (underlined). EMSAs were performed as described previously [START_REF] Andrews | A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells[END_REF]. Briefly, all fragments were labeled with [ 32 P]ATP (NEN Life Science Products-DuPont, Bad Homburg, Germany) using T4 polynucleotide kinase (Fermentas, Lithuania). The radiolabeled probes (0.1-0.5 ng) were incubated with nuclear protein extracts (2.5 µg) for 20 min at room temperature in 20 µl reactions containing 10 mM HEPES, pH 7.9, 100 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 1 mM EGTA and 10% glycerol. The specificity of DNA-binding was determined by competition assays performed using a 25-or 50-fold excess of the unlabeled fragments. Reactions were examined by electrophoresis on non-denaturing 6% polyacrylamide gels prepared in a buffer composed of 45 mM Tris, pH 8.0, 45 mM borate and 1 mM EDTA. After running for 2h, the gels were dried and the radioactive signal visualized using a Bio-Rad Molecular Imager FX and quantitated using Quantity One software (Bio-Rad).

Western blot analysis. 50 µg of nuclear or cytoplasmic proteins, prepared as described previously [START_REF] Roe | Characterization of nuclear protein binding (AP-1, GR, and STAT) in the genetically obese (fa/fa) Zucker rat[END_REF][START_REF] Drapier | Biosynthesis of nitric oxide activates iron regulatory factor in macrophages[END_REF], were separated by electrophoresis on 8% sodium dodecyl sulfatepolyacrylamide gels and transferred to Hybond nitrocellulose membranes (Amersham Biosciences) using Novex® Western Transfer Apparatus (Invitrogen). STAT5a and STAT5b

proteins were detected by incubation of the membranes with anti-STAT5a and anti-STAT5bspecific rabbit antibodies kindly provided by Dr. F. Verdier (INSERM U363, Paris, France) [START_REF] Verdier | A sequence of the CIS gene promoter interacts preferentially with two associated STAT5A dimers: a distinct biochemical difference between STAT5A and STAT5B[END_REF]. Nuclear extracts from UT7 cells (kindly provided by Dr. F. Verdier) overexpressing STAT5a and STAT5b proteins were used as a positive control. Immunoreactive bands were revealed following incubation with a goat anti-rabbit horseradish peroxidase-conjugated secondary IgG (Sigma) using the chemiluminescence ECL-plus kit (Amersham Life Sciences). Quantification was performed relative to β-actin detected using a specific antibody obtained from SIGMA.

Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) analysis of STAT5a

mRNA level. For RT-PCR analysis, total cellular RNA was extracted from RAW 264.7 macrophages (10 7 cells) using TRIZol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Two micrograms of total RNA were used for reverse-transcription with superscript II RNAse H reverse transcriptase (Invitrogen) in a 30 µl reaction incubated at 45ºC for 1 h. Of this reaction, 2 µl were then used for PCR amplification with Platinum Pfx DNA Polymerase (Invitrogen), using oligonucleotide primers specific for STAT5a: forward:

5`-CCGTGGGATGCTATTGACTT-3`, reverse: 5`-ACGTGTTCTGGAGCTGTGTG-3`.

PCR products were analyzed on 2% agarose gels run with TBE buffer and stained with ethidium bromide. The specific band of 169 bp was quantified with a Molecular Imager using Quantity One software (BioRad) and normalized to a 489 bp fragment of the GAPDH housekeeping gene amplified from duplicate samples using the forward primer 5`-GACCACAGTCCATGCCATCAC-3` and the reverse primer 5`-TCCACCACCCTGTTGCTGTAG-3'.

Real-time quantitative RT-PCR. STAT5a mRNA levels in RAW 264.7 cells were measured by real-time quantitative RT-PCR using a Light Cycler (Roche Diagnostics, Mannheim, Germany). The same cDNA templates and primers as those described above were used.

Detection of amplification products was carried out using SYBR Green (Roche) as described previously [START_REF] Simpson | Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR[END_REF]. To confirm amplification specificity, the PCR products from each primer pair were subjected to melting curve analysis and subsequent agarose gel electrophoresis. For data analysis, Light Cycler 3.5 Software was used [START_REF] Simpson | Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR[END_REF]. Quantification was performed relative to GAPDH control reactions.

Statistical analysis.

For statistical evaluation, one-way analysis of variance was applied. For estimation of the significance of difference between arithmetic means, the Scheffe test was used. All calculations were done using Statgraphics Plus 6.0 software. Copyright 2006 Biochemical Society control vector expressing the luciferase under the control of the SV40 promoter was highly expressed in these cells and there was a 60% reduction in luciferase activity following DETA/NO treatment (not shown), as previously reported [START_REF] Fan | Nitric oxide donors inhibit luciferase expression in a promoter-independent fashion[END_REF]. Transient transfections of HuH7 cells with the pGL2-IRP1P0.1 construct containing the minimal promoter fragment showed that the basal luciferase activity produced by this construct was very low and treatment of the cells for 48 hours with a chemical NO donor (DETA/NO, 250 µM, induced a statistically significant 2-fold reduction (p<0.05) in luciferase activity (Fig. 1C). The longer 605 bp fragment from pGL2-IRP1P0.2 construct containing both Sp1 and MRE showed a higher transcriptional activity, with a 3-4 fold increase in luciferase activity as compared to HuH7 pGL2-IRP1P0.1-transfected cells. However, the NO treatment only induced a moderate decrease in luciferase activity. Another construct was made by adding an oligonucleotide with the GAS sequence upstream of pGL2-IRP1P0.2 (Fig. 1B). The basal activity produced by the pGL2-IRP1P0.3 plasmid was the highest among all the analyzed IRP1 promoter-luciferase constructs. Treatment with DETA/NO of pGL2-IRP1P0.3 vector-transfected HuH7 cells revealed a substantial (90%) inhibition of luciferase activity compared with basal conditions (Fig. 1C). Considering that the pGL2-IRP1P0. The mechanism underlying the NO-mediated decrease in STAT/DNA complex formation and inhibition of transcriptional activity of the mouse IRP1 promoter, could involve impaired nuclear translocation of STAT proteins in response to NO. Since the STAT protein family comprises 7 members showing apparently similar binding affinity for the GAS sequence [START_REF] Horvath | STAT proteins and transcriptional responses to extracellular signals[END_REF],

RESULTS

Identification

we focused our attention on the STAT5 proteins whose involvement in signaling pathways inhibited by NO is well recognized [START_REF] Bingisser | Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway[END_REF][START_REF] Mazzoni | Myeloid suppressor lines inhibit T cell responses by an NOdependent mechanism[END_REF]. We measured STAT proteins in cytosolic and nuclear extracts of RAW 264.7 macrophages, using nuclear extracts from UT7 cells overexpressing STAT5a and STAT5b as positive controls. The results showed a marked reduction of STAT5b protein levels in nuclei of NO-producing RAW 264.7 macrophages as compared to control macrophages (Fig. 3, upper panel, lane 2 vs lane 1). However, an even more drastic reduction of both STAT5a and STAT5b proteins, i.e. an almost total absence (Fig. 3. lower panel, lane 2), was observed in the cytosol, which indicates that impaired translocation is not the reason for the reduction in STAT/DNA complex formation. This characteristic pattern of changes in STAT protein levels in these two cellular compartments was specific for NO-producing cells and it was largely reversed when NO synthesis had been abolished by inhibitors of NOS2 (Figs. 3, lane 3).

LPS/IFN-γ-induced reduction of STAT5a expression is not due to mRNA downregulation.

We next examined whether this reduction in the amount of STAT5a protein resulted from a decrease in the level of the corresponding mRNA transcript. Both semi-quantitative RT-PCR (Fig. 4A and4B) and real-time quantitative RT-PCR (Fig. 4C) consistently showed that STAT5a mRNA expression in NO-producing RAW 264.7 macrophages was unchanged relative to control cells or in comparison with macrophages in which NO synthesis was severely reduced. These analyses suggest that the substantial decrease in STAT5a protein content in response to NO is not due to less efficient transcription of the STAT5a gene or decreased stability of STAT5a mRNA.

DISCUSSION

Most studies on the regulation of IRP1 activities have been focused on posttranslational mechanisms underlying the insertion/extrusion of a [4Fe-4S] cluster into/from the IRP1 molecule [START_REF] Bouton | Iron regulatory proteins as NO signal transducers[END_REF][START_REF] Drapier | Redox modulation of iron regulatory proteins by nitric oxide and peroxynitrite[END_REF]. However, we have recently shown that changes in intracellular protein IRP1 levels could account for the observed modifications in its IRE-binding affinity [START_REF] Oliveira | Down-regulation of iron regulatory protein 1 gene expression by nitric oxide[END_REF][START_REF] Lipiński | Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H(2)O(2)[END_REF][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]. It has long been known that in mammals, IRP1 displays highly variable expression patterns in different tissues [START_REF] Müllner | In vivo and in vitro modulation of the mRNA-binding activity of iron-regulatory factor. Tissue distribution and effects of cell proliferation, iron levels and redox state[END_REF]. The biological significance of regulation of IRP1 expression at the protein level are indicated by studies on NO-producing macrophages, in which NO was

shown not only to induce IRP1 RNA binding activity but also to reduce IRP1 protein level [START_REF] Oliveira | Down-regulation of iron regulatory protein 1 gene expression by nitric oxide[END_REF]. The aim of the present study was to investigate the transcriptional regulation of IRP1 in response to NO and to identify regulatory elements within the IRP1 promoter responsible for NO-dependent inhibition of IRP1 expression. Although previous studies have reported no changes in the total amount of immunologically detectable IRP1 following treatment of various cell types with iron sources and iron chelators [START_REF] Tang | Translational repressor activity is equivalent and is quantitatively predicted by in vitro RNA binding for two iron-responsive element-binding proteins, IRP1 and IRP2[END_REF][START_REF] Henderson | Characterization of a second RNAbinding protein in rodents with specificity for iron-responsive elements[END_REF][START_REF] Guo | Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome[END_REF], recent data from closely related mouse lymphoblastic cell lines show a close quantitative correlation between the amount of IRP1 and LIP levels [START_REF] Lipiński | Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H(2)O(2)[END_REF]. Furthermore, it has been demonstrated that hepatic IRP1 protein levels in haemochromatosis patients are altered [START_REF] Neonaki | Down-regulation of liver iron-regulatory protein 1 in haemochromatosis[END_REF]. With this in mind, we also attempted to test the influence of iron on transcriptional regulation of the IRP1 gene. Using bio-informatics software, the mouse IRP1 promoter sequence was screened for putative binding sites for NO-and iron-dependent transcriptional regulators. We identified potential binding sites for Sp1 and STAT proteins, two TFs known to be molecular targets for NO [START_REF] Sellak | Sp1 transcription factor as a molecular target for nitric oxide--and cyclic nucleotide--mediated suppression of cGMP-dependent protein kinase-Ialpha expression in vascular smooth muscle cells[END_REF][START_REF] Bingisser | Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway[END_REF][START_REF] Mazzoni | Myeloid suppressor lines inhibit T cell responses by an NOdependent mechanism[END_REF]. We also found one potential MRE sequence similar to those present in the promoter region of genes encoding metallothionein-II A [START_REF] Palmiter | Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1[END_REF], a heavy-metal binding protein or divalent metal transporter 1 [START_REF] Lee | The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms[END_REF], a mediator of apical iron uptake in duodenal enterocytes and iron transfer from the transferrin receptor endosomal cycle into the cytosol.

To gain further insights into the role of the GAS and Sp1 sequences in NO-mediated repression of the mouse IRP1 gene, we transiently transfected human hepatoma HuH7 cells with constructs containing fragments of the mouse IRP1 promoter fused to the luciferase reporter gene. The minimal 55 bp promoter had a very low basal transcriptional activity, which was reduced by NO production. The mouse IRP1 promoter lacks typical TATA or CCAAT boxes. In many genes lacking a TATA box, a proximally positioned Sp1 site may be critical for basal transcription and can determine the start site [START_REF] Sauerwald | The 5'-flanking region of the synapsin I gene. A G+C-rich, TATA-and CAAT-less, phylogenetically conserved sequence with cell type-specific promoter function[END_REF]. Our results indicate that the proximal Sp1 binding site is of limited importance for basal transcription of IRP1.

However, NO treatment of the transfected cells induced a significant reduction in basal transcriptional activity, and accordingly, nuclear extracts from activated macrophages reduced the DNA binding activity of Sp1. This result is in line with those of previous studies demonstrating inhibition or a lack of DNA binding by native Sp1 derived from different cell types treated with chemical NO donors [START_REF] Sellak | Sp1 transcription factor as a molecular target for nitric oxide--and cyclic nucleotide--mediated suppression of cGMP-dependent protein kinase-Ialpha expression in vascular smooth muscle cells[END_REF][START_REF] Berendji | Zinc finger transcription factors as molecular targets for nitric oxide-mediated immunosuppression: inhibition of IL-2 gene expression in murine lymphocytes[END_REF][START_REF] Wang | A Sp1 binding site of the tumor necrosis factor alpha promoter functions as a nitric oxide response element[END_REF]. It is important to note that the NOmediated loss of Sp1 DNA binding activity may result in either a decrease [START_REF] Sellak | Sp1 transcription factor as a molecular target for nitric oxide--and cyclic nucleotide--mediated suppression of cGMP-dependent protein kinase-Ialpha expression in vascular smooth muscle cells[END_REF][START_REF] Berendji | Zinc finger transcription factors as molecular targets for nitric oxide-mediated immunosuppression: inhibition of IL-2 gene expression in murine lymphocytes[END_REF] or an increase [START_REF] Wang | A Sp1 binding site of the tumor necrosis factor alpha promoter functions as a nitric oxide response element[END_REF] in gene expression depending on whether this factor is acting as an activator or a repressor of transcription. Thus, the results of our EMSA experiments are not conclusive concerning the role of Sp1 in NO-dependent down-regulation of IRP1 gene transcription.

Transfection of HuH7 cells with the second construct, pGL2-IRP1P0.2, containing both Sp1 and MRE sequences, resulted in substantially higher basal transcriptional activity, but no decrease in luciferase activity in response to NO. Accordingly, there was no change in the DNA-binding activity to the MRE sequence in response to fluctuations in cellular iron content or to NO, recently shown to alter LIP level [START_REF] Lipiński | Induction of iron regulatory protein 1 RNA-binding activity by nitric oxide is associated with a concomitant increase in the labile iron pool: implications for DNA damage[END_REF][START_REF] Chenais | Impact of endogenous nitric oxide on microglial cell energy metabolism and labile iron pool[END_REF]. This result agrees with those of previous studies showing that interaction of MTF1 with multiple MREs present in 5'-regulatory regions of several genes is induced by heavy metals other than iron [START_REF] Palmiter | Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1[END_REF]. It is thus possible that the effect of MTF1-mediated modulation of gene transcription may only involve genes containing several copies of the MRE sequence in their promoters [START_REF] Palmiter | Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1[END_REF]. The insensitivity of the binding of MTF-1, a zinc sensor protein [START_REF] Giedroc | Metal response element (MRE)-binding transcription factor-1 (MTF-1): structure, function, and regulation[END_REF], to DNA in response to NO may result from the opposite effects of NO on zinc finger-dependent transcription. Zinc finger disruption by NO may inhibit or activate transcription, depending on the structure of the promoter [START_REF] Kroncke | Nitrosative stress and transcription[END_REF]. NOmediated inhibition of zinc finger-dependent gene transcription [START_REF] Berendji | Zinc finger transcription factors as molecular targets for nitric oxide-mediated immunosuppression: inhibition of IL-2 gene expression in murine lymphocytes[END_REF] may be counteracted by an indirect effect resulting from NO-mediated release of zinc bound to cytosolic lowmolecular weight chelators. The activation of MTF1 DNA binding by such a mechanism has been reported in response to cadmium (Cd) and oxidative stress upon Cd binding or oxidation of metal-coordinating thiolates, respectively [START_REF] Giedroc | Metal response element (MRE)-binding transcription factor-1 (MTF-1): structure, function, and regulation[END_REF].

Functional analysis of the IRP1 promoter using the third construct, pGL2-IRP1P0.3

clearly showed that the GAS sequence plays a major role in both the basal expression level and NO-dependent regulation of the IRP1 gene. It should be stressed that only a 19 bp fragment comprising the GAS sequence from the IRP1 promoter was added to pGL2-IRP1P0.2 in order to generate pGL2-IRP1P0.3. The demonstration that NO produced a 70% decrease in binding of STAT proteins to the GAS sequence located in the IRP1 promoter strongly suggests the involvement of this family of TFs in the regulation of IRP1 gene expression by NO. In agreement with this finding, negative gene regulation in response to NO by STAT5 [START_REF] Bingisser | Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway[END_REF][START_REF] Mazzoni | Myeloid suppressor lines inhibit T cell responses by an NOdependent mechanism[END_REF], and also by STAT1 [START_REF] Giustizieri | Nitric oxide donors suppress chemokine production by keratinocytes in vitro and in vivo[END_REF], has been reported for several cellular functions in various cell types. Taken together, the results of the EMSA experiments and those from the functional activity assays indicate that the GAS sequence is an important regulatory element responsible for down-regulation of IRP1 expression by NO.

The GAS sequence was originally identified as an element required for the rapid transcriptional induction of genes in response to IFN-γ [START_REF] Decker | GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression[END_REF]. The protein complex binding to GAS sequences in IFN-γ-treated cells is a dimer of STAT1, the prototype of the STAT protein family. To date, seven different STATs have been recognized, six of which bind to a GAS element [59]. Because STAT5 proteins are those most frequently shown to take part in nuclear signaling in response to NO [START_REF] Bingisser | Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway[END_REF][START_REF] Mazzoni | Myeloid suppressor lines inhibit T cell responses by an NOdependent mechanism[END_REF], we investigated the role STAT5a and STAT5b in the mechanism underlying NO-dependent down-regulation of IRP1. Although these two STAT5 proteins share a high degree of amino acid sequence identity and show considerable functional overlap they are not functionally redundant [59]. According to a general model for receptor-mediated activation of STAT, there are several cytokine receptor-associated Janus kinases that induce dimerization, nuclear translocation and DNA binding of STAT through tyrosine phosphorylation [START_REF] Horvath | STAT proteins and transcriptional responses to extracellular signals[END_REF]. To identify the mechanism through which NO exerts its repressive effect on IRP1 expression via the GAS sequence, we initially investigated whether it is associated with impaired nuclear translocation of STAT5. Endogenous NO was found to markedly reduce nuclear levels of both STAT5a and STAT5b in RAW 264.7 macrophages. A similar effect was observed when RAW 264.7 cells were incubated for 18 h with DETA/NO (data not shown). Surprisingly, an identical analysis of cytosolic extracts from RAW 264.7 macrophages, activated for NO synthesis, showed an even greater decrease in STAT5a and STAT5b content than that observed in nuclei. The results of these Western Blot analyses indicate an overall decline in STAT5 protein levels rather than one restricted to nuclei. The reduction in the amount of STAT5a at the protein level was not associated with a decrease in the expression or stability of the transcript since STAT5a mRNA levels were not influenced by NO. Taken together, our data on STAT5a mRNA and protein levels in NO-producing macrophages strongly suggest that NO sensitizes STAT5 proteins for accelerated degradation.

This conclusion is supported by the recent description of a proteosome proteolytic pathway for STAT5 and the proposal that this plays a role in down-regulation of STAT5 signalling [START_REF] Wang | A small amphipathic alpha-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5[END_REF]. A hypothetical mechanism for the down-regulation of STAT5 activity by NO may involve nitration of key tyrosine residue(s) in STAT5 molecules and subsequent proteolytic degradation by the ubiquitin-proteosome pathway as has been described for other proteins [START_REF] Schopfer | NO-dependent protein nitration: a cell signaling event or an oxidative inflammatory response?[END_REF].

The findings of this study permit us to outline a plausible regulatory loop between STAT family proteins, nitric oxide and IRP1. Transcription factors from the STAT family participate in signal transduction initiated by IFN-γ leading to the activation of NOS2 expression and subsequent high-output of NO [START_REF] Blanchette | Signalling events involved in interferon-gamma-inducible macrophage nitric oxide generation[END_REF]. The action of NO rapidly generates the RNA binding form of IRP1 [START_REF] Drapier | Redox modulation of iron regulatory proteins by nitric oxide and peroxynitrite[END_REF], which downregulates ferritin and upregulates transferrin receptor via a posttranscriptional mechanism, thus contributing to the expansion of the catalytically active free iron pool. On the other hand, NO would promote degradation of STAT5 proteins which inhibits expression of the IRP1 gene at transcriptional level. The biological importance of this regulation may be in minimizing the NO-dependent reaction that boosts RNA binding by IRP1 and in consequence, attenuates the NO-induced rise in the labile iron pool, a major factor in increasing the likelihood of oxidative injury.

Finally, a question arises as to the possible role of STAT5-driven regulation of IRP1 described here, in the cross-talk between NO and iron in cells. It is conceivable to propose that iron released from iron-containing proteins upon NO enters LIP and transits through the cytosolic compartment before being transported across the cellular membrane as nitrosyl-iron complexes by multidrug resistance-associated protein 1, as suggested recently [START_REF] Watts | Nitrogen monoxide (NO)mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1[END_REF]. This socalled transient iron may be easily taken up by ferritin, a main regulator of LIP levels [START_REF] Picard | Role of ferritin in the control of the labile iron pool in murine erythroleukemia cells[END_REF]. By decreasing the amount of IRP1 via STAT5, NO minimizes its potential to inhibit ferritin expression at post-transcriptional level, and may thus maintain and saveguard the capacity of this protein to sequester potentially harmful free iron.

  Materials. Dulbecco's modified Eagle's medium (DMEM) was obtained from GIBCO/BRL. Low endotoxin fetal calf serum (FCS), ferric ammonium citrate (FAC), desferrioxamine ® (DFO) and lipopolysaccharide (LPS) from Escherichia coli (serotype O55:B5) were purchased from Sigma (St. Louis, MO, USA). Murine recombinant interferon-γ (IFN-γ) was from RandD Systems, UK. N-[3-(aminomethyl)benzoyl]acetamide (1400W), L-N Gmonomethyl-L-arginine (L-NMMA) and diethylentriamine NONOate (DETA/NO) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Restriction and modification enzymes were from Invitrogen (Carlsbad, CA, USA). All oligonucleotides were synthesized by Invitrogen. Competent E. coli DH5α™ cells, Ortho-Nitrophenyl-βD-galactopyranoside (ONPG) and lipofectamine were from Invitrogen. Luciferin was from Euromedex, France. All other chemicals were obtained from Sigma. Cell Culture and Treatment. RAW 264.7 murine macrophages, a cell line established from a tumor induced by Abelson murine leukemia virus, were obtained from the American Type Culture Collection (Rockville MD, USA). Cells were cultured in DMEM containing 5% FCS, and gentamycin (50 µg/ml) in 100 cm 2 plastic culture flasks (NUNC, Denmark) in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. For nitric oxide synthase 2 (NOS2) induction, RAW 264.7 macrophages were stimulated for 16 h with 20 U/ml IFNγ and 50 ng/ml LPS. In other experiments these cells were incubated with 250 µM DFO and/or 100 µM FAC for 24 h. The human hepatoma cell line HuH7 was maintained in DMEM supplemented with 10% FCS, 50 U/ml penicillin and 50 µg/ml streptomycin. Nitrite Measurement. Nitric oxide (NO) production was determined by quantifying nitrite, the stable end product of NO, spectrophotometrically by a colorimetric assay. Briefly, 200 µl of sample medium were reacted with 800 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine), and the absorbance was measured at 543 nm. Sodium nitrite was used as a standard. Plasmid constructs. Two fragments from the IRP1 promoter region were PCR amplified to +73 bp) and the same reverse primer promIRP1R 3'-CCGCTCGAGCCGACTCCGCGCTCCTCCCACTGA-5'. The two PCR fragments of respectively 195 bp and 605 bp were digested with Xho I/Mlu I and ligated to the Xho I/Mlu I-cleaved pGL2-basic vector (Promega, Madison, WI) to create plasmids pGL2-IRP1P0.1 and pGL2-IRP1P0.2 in which the IRP1 promoter sequences were transcriptionally fused in the correct orientation to the luciferase reporter gene. To generate a third construct, pGL2-IRP1P0.3, a double-stranded oligonucleotide containing the upstream GAS sequence from the IRP1 promoter (bold) as well as 5' KpnI (underlined) and a 3' MluI (underlined) restriction site 5'-CCGGGTACCAGGCATTTCTAGGAAAATAAGAACGCGTGCC-3' was inserted Biochemical Journal Immediate Publication. Published on 4 Aug 2006 as manuscript BJ20060623 Copyright 2006 Biochemical Society into the pGL2-IRP1P0.2 plasmid following Mlu I/Kpn I digestion to place it upstream of the 605 bp fragment. The orientation and the integrity of all plasmids were verified by sequencing. Fig. 1B shows a schematic representation of the luciferase reporter constructs used in this study.

  of putative binding sites for transcription factors(s) regulated by NO and iron in the IRP1 promoter.The -2790 to +90 bp fragment of the mouse IRP1 gene (GenBank accession no. AJ427344) was searched for potential binding sites for transcription factors(s) regulated by NO and/or changes in intracellular iron level using the Transcription Element Search System software (TESS; http://www.cbil.upenn.edu/tess/) and TRANSFAC database (http://www.generegulation.com/pub/databases.html#transfac). We found the following conserved motifs: one Sp1 sequence, 55 bp away from the transcription start site, one MRE sequence, 128 bp away from the transcription start site and one GAS motif, a potential binding site for STAT family proteins, 1345 bp upstream of the transcription start site (Fig.1A). All identified sequences are also present in the human IRP1 promoter (GenBank accession no. AL161783) at approximately the same distances from the transcription start site (Fig1A).Predominant role of the GAS sequence in the inhibition of mouse IRP1 promoter activity by nitric oxide in transfected HuH7 cells.To perform functional studies of the IRP1 promoter and analyse its regulation by NO, three luciferase-based reporter constructs containing sub-fragments of the IRP1 promoter sequence, with sizes ranging from 195 to 624 bp (Fig.1B) were transiently transfected into HuH7 cells and the cells were grown for 48 hours with or without DETA/NO as a NO donor. The pGL3 Biochemical Journal Immediate Publication. Published on 4 Aug 2006 as manuscript BJ20060623

  3 construct was generated by the addition, upstream of the 605 bp fragment in pGL2-IRP1P0.2, of a short 19 bp fragment comprising the upstream GAS sequence from the IRP1 promoter, this result suggests the effective and predominant role of the STAT binding site in both the basal transcriptional activity and the NO-dependent regulation of the mouse IRP1 promoter. Nitric oxide inhibits Sp1 and STAT DNA binding, but has no effect on MTF1 DNA binding. Since the Sp1 and the STAT-binding sites appear to be functional and play a role in NOmediated down-regulation of IRP1 promoter we decided to study the effect of NO on the DNA binding activity of Sp1 and STATs in RAW 264.7 macrophage nuclear extracts. We performed EMSAs using short DNA probes comprising the Sp1 binding site or the GAS sequence of the mouse IRP1 promoter. The specificity of the constitutive DNA-binding reactions (Figs. 2A and 2B, lanes 1) was confirmed by competition with a 50-fold (Figs. 2A and 2B, lanes 4) and a 25-fold (Figs. 2A and 2B, lanes 5) molar excess of the unlabelled probes. NO production was stimulated by treating the cells with IFN-γ and LPS and was followed by measuring nitrite (a stable oxidation product of NO) released in the culture medium (as indicated below Figs. 2A and 2B). The formation of Sp1/DNA (Fig. 2A, lane 2) and STAT/DNA (Fig 2B, lane 2) complexes in reactions including nuclear extracts from NOproducing RAW 264.7 cells was markedly reduced as compared with those using extracts from control cells (Figs 2A and2B, lanes 1). This decrease was 65 and 47%, respectively, as assessed by densitometric analysis. The effect of NO was largely reversed when EMSAs were performed using nuclear extracts from stimulated macrophages treated with 1400W and L-NMMA, two NO synthase 2 inhibitors (Figs 2A and 2B, lanes 3). EMSA was also used to visualize nuclear protein complexes with the MRE sequence overlapping the -128 to -134 region of the IRP1 promoter in NO-producing macrophages (Fig.2C). The specificity of the basal MTF1 binding to MRE in control RAW 264.7 macrophages (Fig.2C, lane 1) was confirmed by competition with a 50-fold excess (Fig.2C, lane 2) of the unlabelled MRE probe. NO has been shown to affect labile iron pool (LIP) levels in various cell types[START_REF] Shin | Nitric oxide attenuates vascular smooth muscle cell activation by interferon-gamma. The role of constitutive NF-kappa B activity[END_REF][START_REF] Pilz | Nitric oxide and cGMP analogs activate transcription from AP-1-responsive promoters in mammalian cells[END_REF], and thus may indirectly influence the DNA binding activity of MTF1, a TF known to bind MRE and to be regulated by heavy metals[START_REF] Radtke | Functional domains of the heavy metal-responsive transcription regulator MTF-1[END_REF]. However, we detected no changes in MTF1 binding to MRE in RAW macrophages using nuclear extracts from cells stimulated for NO synthesis with IFN-γ/LPS (Fig.2C, lane 3). Similarly, in macrophages incubated either with ferric ammonium citrate (FAC) (Fig.2C, lane 4) or DFO, an iron chelator (Fig.2C, lane 5) the level of MTF1/DNA complexes was unaffected compared with control cells, clearly indicating that fluctuations in intracellular iron level had no effect on MTF1 DNA binding.Nitric oxide decreases STAT5a and STAT5b protein levels in the cytosol and nuclei of NO-producing macrophages.

Figure 1 :

 1 Figure 1: Functional studies of the IRP1 promoter region. A, The location of putative binding sites for the transcription factors regulated by NO and/or changes in intracellular iron level in the mouse IRP1 promoter. The -2790 to +90 bp fragment of the mouse IRP1 gene (GenBank accession no. AJ427344) was analyzed using Transcription Element Search System (TESS) software and the TRANSFAC database. The indicated sequences located at positions -63 to -55 bp, -134 to -128 bp, and -1354 to -1345 bp upstream of the transcription initiation site, represent three potential cis-acting regulatory elements corresponding to Sp1, MRE, and the GAS sequence, respectively. The consensus sequences and the position of the above-mentioned putative binding sites in human IRP1 gene sequence (AL161783) are indicated. B, Structure of reporter gene constructs used in transient transfection assays. All constructs were prepared using the pGL2-basic vector.Two overlapping fragments of the mouse IRP1 promoter sequence extending from -122 to +

Figure 2 .

 2 Figure 2. Effect of endogenous nitric oxide on Sp1, STAT, and MTF1 DNA binding activity in mouse 264.7 macrophages. Two µg of nuclear protein and a molar excess of [ 32 P] -labeled probes containing the Sp1 (A), GAS (B) or MRE (C) sequences from the mouse IRP1 promoter were used in EMSA analysis as described in Materials and Methods. Cultured RAW 264.7 macrophages were stimulated for 16 h with 20 units/ml IFN-γ plus 50 ng/ml LPS in the presence or absence of 1 mM L-NMMA and 25 µM 1400W (A and B). NO production was measured by assaying nitrites using Griess method in culture supernatants of stimulated and unstimulated cells. RAW 264.7 macrophages were incubated with 250 µM DFO and/or 100 µM FAC for 24 h (C). To check the specificity of the DNA-binding, unlabeled competitor fragments were added to reactions at 25-and 50-fold molar excess over the labeled probes. Reaction products were separated by electrophoresis on non-denaturing 6% polyacrylamide gels. The experiments were performed at least three times, and representative gels are shown. The positions of the DNA-protein complexes and the free probes are

Figure 3 .

 3 Figure 3. Reduced STAT5a and STAT5b protein levels in NO-producing RAW 274.7 macrophages. RAW 264.7 macrophages were stimulated for 16 h with 20 units/ml IFN-γ plus 50 ng/ml LPS in the presence or absence of 1 mM L-NMMA and 25 µM 1400W. NO production was measured by assaying nitrite in the culture medium. STAT5a and STAT5b levels were assessed in nuclear (upper panel) and cytosolic (lower panel) extracts by Western blotting as described in Materials and Methods. To assist identification of STAT5a and STAT5b bands, nuclear extracts from UT7 cells overexpressing STAT5a and STAT5b proteins (a generous gift from Dr. Frederic Verdier) were electrophoresed on the same gels. Each blot is representative of three blots showing similar results.

Figure 4 .

 4 Figure 4. Analysis of STAT5a mRNA abundance in NO-producing RAW 264.7 macrophages by RT-PCR. RAW 264.7 macrophages were stimulated for 16 h with 20 units/ml IFN-γ plus 50 ng/ml LPS in the presence or absence of 1 mM L-NMMA and 25 µM 1400W. NO production was measured by assaying nitrite in the culture medium. RNA was isolated from harvested cells and used for semi-quantitative RT-PCR as described in Materials and Methods. The GAPDH amplicon was used as a control for the amount of cDNA used in the PCR reactions. The experiments were performed 3 times, and a representative result is shown (A). PCR product bands were quantified using a Molecular Imager and the means ± SD are plotted in arbitrary units (n=3) (B). STAT5a mRNA abundance in RAW 264.7 macrophages was measured by real-time RT-PCR (C). Each column represents the mean (± SD) of 3 amplification reactions performed on a single cDNA sample reverse transcribed from RNA prepared from cells from 3 independent experiments. The level of STAT5b mRNA in control macrophages was assigned as the 100% value.

Figure 1

 1 Figure 1.

ACKNOWLEDGMENTS:

This work was supported by a NATO Collaborative Linkage Grant (PST.CGL 979519). We thank John Gittins for critical reading the manuscript.