Intracellular interleukin-1 receptor antagonist type 1 antagonizes the stimulatory effect of interleukin-1 alpha precursor on cell motility

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Reference

Intracellular interleukin-1 receptor antagonist type 1 antagonizes the stimulatory effect of interleukin-1 alpha precursor on cell motility

MERHI-SOUSSI, Faten, et al.

Abstract

Interleukin (IL)-1alpha, a proinflammatory cytokine, is produced as a 33 kDa protein precursor (preIL-1alpha) which is cleaved to generate the 17 kDa C-terminal mature IL-1alpha (mIL-1alpha) and the 16kDa N-terminal IL-1alpha propiece (NIL-1alpha). The biological effect of IL-1alpha is regulated by the IL-1 receptor antagonist (IL-1Ra), its naturally occurring inhibitor. Four different isoforms of the IL-1Ra have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Whether the icIL-1Ra1 isoform can antagonize some of the biological effects of intracellular IL-1alpha is still unknown. The aim of this study is to investigate effects of preIL-1alpha and icIL-1Ra1 on cell motility in stably transfected ECV304 cells. We show that expression of preIL-1alpha in ECV304 cells significantly increases cell motility. Furthermore, transfection with NIL-1alpha propiece also increases cell motility whereas this stimulatory effect was not observed by addition of exogenous mIL-1alpha, suggesting an intracellular effect of preIL-1alpha mediated by NIL-1alpha propiece.

Co-transfection of ECV304 cells with [...]

MERHI-SOUSSI, Faten, et al . Intracellular interleukin-1 receptor antagonist type 1 antagonizes the stimulatory effect of interleukin-1 alpha precursor on cell motility. Cytokine , 2005, vol. 32, no. 3-4, p. 163-70

PMID : 16246569

DOI : 10.1016/j.cyto.2005.09.004

Available at:

http://archive-ouverte.unige.ch/unige:26033

Disclaimer: layout of this document may differ from the published version.

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Intracellular interleukin-1 receptor antagonist type 1 antagonizes the stimulatory effect of interleukin-1 a precursor on cell motility

Faten Merhi-Soussi

^a,b

, Marina Berti

^a,b

, Bernhard Wehrle-Haller

^b

, Cem Gabay

^a,b,

*

aDivision of Rheumatology, Department of Internal Medicine, University Hospital of Geneva, 26 avenue de Beau-se´jour, 1211 Geneva 14, Switzerland

bDepartment of Pathology and Immunology, University of Geneva, School of Medicine, Geneva, Switzerland Received 6 April 2005; received in revised form 6 September 2005; accepted 9 September 2005

Abstract

Interleukin (IL)-1a, a proinflammatory cytokine, is produced as a 33 kDa protein precursor (preIL-1a) which is cleaved to generate the 17 kDa C-terminal mature IL-1a(mIL-1a) and the 16 kDa N-terminal IL-1apropiece (NIL-1a). The biological effect of IL-1ais regulated by the IL-1 receptor antagonist (IL-1Ra), its naturally occurring inhibitor. Four different isoforms of the IL-1Ra have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Whether the icIL-1Ra1 isoform can antagonize some of the biological effects of intracellular IL-1ais still unknown. The aim of this study is to investigate effects of preIL-1aand icIL-1Ra1 on cell motility in stably transfected ECV304 cells. We show that expression of preIL-1ain ECV304 cells significantly increases cell motility. Furthermore, transfection with NIL-1a propiece also increases cell motility whereas this stimulatory effect was not observed by addition of exogenous mIL-1a, suggesting an intracellular effect of preIL-1amediated by NIL-1apropiece. Co-transfection of ECV304 cells with icIL-1Ra1 completely antagonizes the stimulatory effect of preIL-1aand NIL-1apropiece on cell motility. In conclusion, NIL-1apropiece increases ECV304 cell motility and icIL-1Ra1 exerts intracellular functions regulating this stimulatory effect.

Keywords:Cell motility; ECV304 cell line; Interleukin-1aprecursor; Interleukin-1 receptor antagonist; N terminal interleukin-1apropiece

1. Introduction

The cytokine interleukin (IL)-1 is a critical mediator of the immune and inflammatory responses. IL-1ais produced as an intracellular precursor protein of 33 kDa (preIL-1a) which is cleaved, by membrane associated calcium sensitive cystein proteases called calpains, into the 17 kDa mature carboxyl

terminus IL-1a (mIL-1a) and the 16 kDa N-terminal IL-1a propiece (NIL-1a) (reviewed in [1]). Both preIL-1a and mIL-1aare biologically active as exogenous cytokines in vitro [2]. Because of the lack of a leader peptide, preIL-1aremains mostly intracellular [1,2]. PreIL-1a contains a functional nuclear localization sequence (NLS) within the N-terminal propiece domain [2,3], thus in transfected human vascular smooth muscle cells (VSMC) and ECV304 cell line, preIL- 1ais located in the nucleus, whereas mIL-1aremains in the cytosol[4,5]. PreIL-1aregulates the proliferation and the migration of transfected ECV304 cells[5,6]. Recently, a nuclear target of preIL-1awas identified, which was associated with a novel function of preIL-1ain transcriptional control[7]. Fur- thermore, NIL-1ais biologically active by itself behaving as a nuclear oncoprotein when expressed in rat mesangial cells [8] and inducing apoptosis in numerous human tumor cell lines and likely operates through a mechanism involving the

Abbreviations:IL-1a, interleukin-1a; preIL-1a, precursor IL-1a; NIL-1a, N terminal IL-1a propiece; mIL-1a, mature IL-1a; IL-1Ra, interleukin-1 receptor antagonist; icIL-1Ra1, intracellular interleukin-1 receptor antagonist type 1; sIL-1Ra, secreted interleukin-1 receptor antagonist; NLS, nuclear localization sequence.

* Corresponding author. Division of Rheumatology, Department of Internal Medicine, University Hospital of Geneva, 26 avenue de Beau-se´jour, 1211 Geneva 14, Switzerland. Tel.:C41 22 382 3500; fax:C41 22 382 3509.

E-mail address:cem.gabay@hcuge.ch(C. Gabay).

doi:10.1016/j.cyto.2005.09.004

Cytokine 32 (2005) 163e170

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RNA processing apparatus and the alternate splicing of apoptosis regulatory proteins [9]. NIL-1awas also found to be specifically associated with the novel protein HAX-1 that modulates apoptosis and cytoskeletal functions[10].

IL-1 receptor antagonist (IL-1Ra) is a member of the IL-1 family that binds to IL-1 receptors, but does not induce intracellular signaling. Four different IL-1Ra peptides are produced from the same gene (IL-1RN) by the use of alternative first exons, mRNA splicing and alternative translation initiation.

One isoform comprises a leader sequence and is secreted (sIL-1Ra). The three other isoforms lack the leader peptide and remain intracellular (icIL-1Ra1, 2, 3)[11]. The transcription of sIL-1Ra and icIL-1Ra1 is regulated by different promoter regions [12]. icIL-1Ra1 binds to IL-1 receptors with the same affinity as sIL-1Ra and inhibits the biologic effects of IL-1 in vitro[13]. Although icIL-1Ra isoforms may be released from dying cells or under certain conditions and function as IL-1 inhibitors, other important regulatory functions inside cells may exist. Studies performed in malignant human ovarian epithelial cells indicated that the expression of icIL- 1Ra1 inhibited mature IL-1a-induced IL-8 and GRO expression by decreasing their mRNA stability [14]. Furthermore, overexpression of icIL-1Ra1 in Caco-2 intestinal epithelial cells inhibited the production of IL-6 and IL-8 in response to IL-1 and this effect was apparently related to an intracellular activity[15].

Whether the icIL-1Ra1 isoform can antagonize some of the biological effects of preIL-1a is still unknown. We recently observed that co-expression of icIL-1Ra1 reverses the production of IL-6 induced by preIL-1a in the stably transfected human SaOS-2 osteosarcoma cell line. In contrast, the growth inhibitory effect induced by preIL-1awas not reversed in icIL- 1Ra1 co-transfected cells, indicating that intracellular effects of preIL-1a are not necessarily sensitive to inhibition by icIL-1Ra1[16]. Here we study the effect of preIL-1aon cell motility in stably transfected ECV304 cells and investigate the possibility that icIL-1Ra1 may function as an intracellular antagonist of preIL-1a activity. We show that expression of preIL-1aor NIL-1apropiece significantly increases cell motility and co-transfection with icIL-1Ra1 completely reverses this stimulatory effect.

2. Materials and methods 2.1. Reagents

M-199 medium, DMEM (4.5 g/L glucose), PBS, fetal bovine serum (FBS), penicillin-streptomycin and trypsine EDTA solution were all purchased from Invitrogen (Basel, Switzerland). Gelatin type B, Nonidet P-40, Glycerol and Tween-20 were purchased from Sigma Fluka Chemie AG (Buchs, Switzerland).

2.2. Plasmid construction

The pcDNA3.1 plasmid (Invitrogen) containing the full length coding region of human preIL-1a(GenBank accession

number NM_000575), inserted in BamHI/XhoI sites of the pcDNA3, and the pHook plasmid containing the full length coding region of human icIL-1Ra1 (GenBank accession number NM_000577), were kindly provided by Dr William P. Arend (University of Colorado Health Sciences Center, Denver, CO). The coding region of NIL-1a was amplified by PCR from the plasmid pcDNA-preIL-1a. The primers used were a forward T7 promoter/priming sequence (TAA- TACGACTCACTATAGGG) from the pcDNA plasmid, in front of a multiple cloning sites containing the BamHI site, and a reverse primer adding aXhoI site after the end of the NIL-1a coding sequence (5#-GGCCGCTCGAGCTACC- TAGGCTTGATGATTTCTTCC-3#). The Flag-tagged NIL-1a (NIL-1a-Flag) was also generated by a PCR amplification of pcDNA-preIL-1a cDNA. The 3# primer encodes an XhoI site followed by an eight amino acid Flag peptide tag (DYKDDDDK) and by NIL-1a coding sequence (5#- GGCCGCTCGAGCTACTTGTCGTCGTCGTCCTTGTAGTC- CCTAGGCTTGATGATTTCTTC-3#). We also used the T₇ promoter as 5# primer. The NIL-1a and NIL-1a-Flag PCR products were purified (Qiagen, Basel, Switzerland), digested withBamHI andXhoI and inserted into pcDNA3 to obtain the expression vectors pcDNA3-NIL-1a and pcDNA3-NIL- 1a-Flag. NIL-1a and NIL-1a-Flag inserts were sequenced by automated DNA sequencing (dideoxynucleotide chain termination method) using the T7primer.

2.3. Cell culture and transfection

ECV304 cells were grown to subconfluence in T-75 Flasks (Falcon) coated with 2% gelatin in M-199 culture medium containing 10% heat inactivated FBS, 100mg/ml streptomycin and 100 U/ml penicillin. The A431 human epidermoid carcinoma cells were grown in DMEM containing 10% heat inactivated FBS (v/v). All cell cultures were conducted in a humid atmosphere containing 5% CO2, at 37 ^!C. ECV304 cells were stably transfected with 10mg of either the empty pcDNA3 vector, or pcDNA3 containing preIL-1a, or NIL-1a or NIL- 1a-Flag, using the calcium phosphate procedure (Promega, Wallisellen, Switzerland). Individual clones were selected with 1.2 mg/ml G418 (Promega). We obtained clones of cells expressing 0.1e1 ng preIL-1a/10⁶ cells and selected clones expressing 1 ng preIL-1a/10⁶cells for the study. To determine the role of icIL-1Ra1, cells transfected with empty pcDNA3 or the expression vector pcDNA3-preIL-1a or pcDNA3-NIL- 1a-Flag were co-transfected with 10mg of either the empty pHook vector or pHook containing icIL-1Ra1 and then, selected with 100mg/ml zeocine (Invitrogen) and 1.2 mg/ml G418. We obtained the following double transfected clones: ECV-vectors (pcDNA3/pHook), ECV-icIL-1Ra1 (pcDNA3/pHook-icIL-1Ra1), ECV-preIL-1a (pcDNA3-preIL-1a/pHook), ECV-preIL-1a/

icIL-1Ra1 (pcDNA3-preIL-1a/pHook-icIL-1Ra1), ECV-NIL- 1a-Flag (pcDNA3-NIL-1a-Flag/pHook), ECV-NIL-1a-Flag/

icIL-1Ra1 (pcDNA3-NIL-1a-Flag/pHook-icIL-1Ra1). The expression of NIL-1a, NIL-1a-Flag, preIL-1a and icIL-1Ra1 was verified by RTePCR (data not shown) and by ELISA.

164 F. Merhi-Soussi et al. / Cytokine 32 (2005) 163e170

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2.4. ELISA for IL-1aand IL-1Ra in transfected ECV304 cells

Confluent transfected ECV304 cells in 24-well gelatin- coated plates were incubated for 72 h in complete medium.

Triplicate wells were set up for each assay condition. Superna- tants were harvested and confluent cells were lysed in ice cold PBS containing 1% Nonidet P-40. Culture supernatants and cell lysates were stored at "20 ^!C. preIL-1a and icIL-1Ra1 concentrations in culture supernatants and cell lysates were measured using a commercial Duo Set ELISA Development System from R & D Systems (Abington, UK). The immunoas- say for IL-1arecognizes both pre- and mIL-1a.

2.5. Cell migration assay

The migration of transfected ECV304 cells was analyzed in Transwell chambers (Costar), as described[17]. Briefly, nucleopore filters (8mm pore size) were coated with 2% gelatin and placed on the top of the lower chambers, which contained serum-free media. Cells (6!10⁴), in serum-free media, were added to the upper chambers. The lower chambers did not contain any chemoattractant. After 16 h of incubation at 37^!C, non migrating cells on the top of the filters were re- moved with a cotton swab, the filters were fixed with methanol and stained with 0.5% crystal violet containing 25% methanol for 60 min. The migrating cells on the lower surface of the membrane were counted in nine randomly chosen fields (150!) per well in triplicate wells. In some wells, recombinant mIL-1a(50 pg/ml) was added exogenously to the upper chamber containing ECV-vectors.

2.6. Video microscopy analysis of two-dimensional cell migration

Time-lapse studies were performed as described previously [18]. Transfected ECV304 cells were plated in serum free media on glass coverslips previously coated with 2% gelatin.

Live cells were observed under an inverted microscope (Zeiss). Time-lapse pictures were acquired with a Hamamatsu C4742-9510 digital charge-coupled device camera (Hama- matsu Photonics, Shizuoka, Japan) controlled by the Openlab software (Improvision, Oxford, UK). Images were recorded at 5-min intervals for 16 h. For time-lapse recordings, cells were kept at a constant temperature of 37^!C and 5% CO2. The average speed of cell migration (mm/h) was calculated using the Openlab software.

2.7. Cell extract preparation

Confluent monolayers of transfected ECV304 cells and of A431 cells in 10-cm dishes were used to prepare cytosol and nuclear fractions, as described [19]. Briefly, confluent cells were washed in cold PBS and lysed in E buffer (0.3% Nonidet P-40, 10 mM Tris, pH 8.0, 60 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM PMSF), by 5-min incubation on ice.

Nuclei were pelleted by spinning for 5 min at 2500 rpm at

4 ^!C in an Eppendorf microcentrifuge, and the supernatants were collected as cytoplasmic extracts. Nuclei were washed in E buffer without Nonidet P-40 and resuspended to 45ml of C buffer (20 mM HEPES, pH 8, 0.2 mM EDTA, 2 mM EGTA, 2 mM DTT, 20% glycerol, 1 mM PMSF, 2mg/ml apro- tinin, 2mg/ml leupeptin). NaCl was added to a final concentra- tion of 0.4 M, and the nuclei were gently shaken for 10 min and centrifuged at 14,000 rpm at 4 ^!C. The nuclear and cytoplasmic extracts were stored at"80 ^!C until measurements of IL-1Ra levels by ELISA were performed.

2.8. Statistical analysis

Data were compared by ANOVA/Scheffe test. A difference between experimental groups was considered significant when thepvalue was less than 0.05. Data are presented as meanG S.E.M. of triplicates from a representative experiment.

3. Results

3.1. Expression of IL-1aand IL-1Ra in transfected ECV304 cells

To investigate the effects of intracellular IL-1aand IL-1Ra on cell motility, we stably co-transfected ECV304 cells with the pcDNA-preIL-1a expression vector or empty pcDNA3 and with pHook-icIL-1Ra1 or empty pHook. The following double transfected cells were obtained: cells transfected with empty pcDNA3/pHook vectors as a control (ECV-vectors), with pcDNA/pHook-icIL-1Ra1 (ECV-icIL-1Ra1), with pcDNA- preIL-1a/pHook (ECV-preIL-1a), with pcDNA-preIL-1aand pHook-icIL-1Ra1 (ECV-preIL-1a/icIL-1Ra1). IL-1a and IL- 1Ra expression levels were measured in cell lysates and culture supernatants (Fig. 1). The levels of IL-1a in lysates of ECV-preIL-1a and ECV-preIL-1a/icIL-1Ra1 were 1.05G 0.24 and 1.17G0.44 ng/10⁶ cells, respectively (Fig. 1A).

Low levels of preIL-1a were released into the supernatants of ECV-preIL-1aand ECV-preIL-1a/icIL-1Ra1 after 72 h of incubation (0.16G0.016 and 0.26G0.015 ng/10⁶ cells, respectively) (Fig. 1A). IL-1a expression was not detectable in lysates or supernatants of cells transfected with empty vectors or transfected with icIL-1Ra1 alone. The concentrations of IL-1Ra in cell lysates of ECV-icIL-1Ra1 and ECV-preIL- 1a/icIL-1Ra1 were 28G2.6 and 33.4G3.37 ng/10⁶ cells, respectively (Fig. 1B). Small amounts of IL-1Ra were detected in supernatants of ECV-icIL-1Ra1 and ECV-preIL-1a/icIL- 1Ra1 after 72 h of incubation (2.5G0.12 and 3.2G 0.11 ng/10⁶cells, respectively) (Fig. 1B). IL-1Ra was not detectable, neither in lysates nor in supernatants of transfected ECV-vectors and ECV-preIL-1acells. The procedure of double transfection of preIL-1a/icIL-1Ra1 was repeated 3 times.

In these transfections, clones or bulks of cells expressing levels of IL-1aand/or IL-1Ra comparable to levels presented inFig. 1were selected for the following studies.

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3.2. preIL-1astimulates ECV304 cell motility and icIL-1Ra1 completely antagonizes the preIL-1aeffect

We stably co-transfected ECV304 cells with pcDNA-preIL- 1a or empty pcDNA3 and with pHook-icIL-1Ra1 or empty pHook and analyzed a potential effect of preIL-1a and/or icIL-1Ra1 on the motility of double-transfected cells using Transwell chambers. Nucleopore filters were coated with gelatin and placed on the top of the lower chambers, which contained serum-free media. Transfected cells, in serum free media, were added to the upper chambers. The lower chambers did not contain any chemoattractant. In these conditions, the cells move spontaneously through the pores and thus, the number of migrating cells reflects the spontaneous motility of these cells. As shown inFig. 2, expression of preIL-1asig- nificantly enhanced the number of migrating cells (63G4 cells/field) as compared to cells transfected with empty vectors

(37G4 cells/field). To assess whether the stimulatory effect of preIL-1a on cell motility was a result of its intracellular activity or due to its partial release into the supernatant, we examined the ability of exogenous recombinant mIL-1a (50 pg/ml) to regulate the motility of ECV-vectors. The addition of recombinant mIL-1a (50 pg/ml) did not modify the number of migrating ECV-vectors (Fig. 2). Higher levels of mIL-1a (up to 1000 pg/ml) were also devoid of effect on cell motility (data not shown), suggesting that preIL-1a enhanced ECV304 cell motility by a mechanism independent of cell surface receptor binding.

Next, we investigated the potential effect of icIL-1Ra1 on cell motility. ECV-icIL-1Ra1 exhibited a migratory response comparable to control cells (38G2 cells/field), indicating that ECV304 cell motility was not affected by icIL-1Ra1 alone. However, co-expression of icIL-1Ra1 in preIL-1atrans- fected cells completely reversed the stimulatory effect of preIL-1a on cell motility (34G5 cells/field). The same results were obtained using clones as well as bulk of transfected cells from three different double transfections. Together these results suggest that preIL-1aincreases ECV304 cell motility, likely via an intracellular mechanism and that icIL-1Ra1 completely antagonizes this stimulatory effect.

3.3. The stimulatory effect of preIL-1aon cell motility was mediated by its N-terminal IL-1apropiece

To investigate whether the effect of preIL-1aon ECV304 cell motility was mediated intracellularly by its N-terminal propiece, we co-transfected ECV304 cells with pcDNA-NIL- 1a-Flag and empty pHook. NIL-1a-Flag transfected cells showed an enhanced migratory response (127G11 cells/

field) as compared to cells transfected with empty vectors (39G4 cells/field) (Fig. 3A). To determine whether the stimulatory effect of NIL-1aon cell motility was due to its activity

Fig. 1. Analysis of cell-associated and supernatant levels of IL-1aand IL-1Ra in stably transfected ECV304 cells. IL-1a(A) and IL-1Ra (B) concentrations in culture supernatants and cell lysates of ECV304 cells transfected with preIL-1aand/or icIL-1Ra1 (ECV-preIL-1a; ECV-icIL-1Ra1; ECV-preIL-1a/

icIL-1Ra1) were measured after 72 h incubation using a commercial Duo Set ELISA, as described in Section2. Cytokine concentrations in supernatants and cell lysates are indicated as ng/10⁶cells. Expression levels of IL-aand IL-1Ra presented in this figure are representative of the levels from three independent double transfections.

Fig. 2. Migratory response of preIL-1aand/or icIL-1Ra1 transfected ECV304 cells. The motility of empty vectors, preIL-1a, icIL-1Ra1, and preIL-1a/icIL- 1Ra1 transfected ECV cells was analyzed using transwell chambers (see Section2). In some wells, recombinant mIL-1a(50 pg/ml) was added to the upper chamber containing ECV-vectors. The migrating cells were counted in nine randomly chosen fields (150!) per well in triplicate wells. This figure is representative of six experiments from three different double transfections.

*p!0.05 vs. ECV-vectors;^yp!0.05 vs. ECV-preIL-1a.

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inside cells and not to its release or to the secretion of other mediators out of the cells, we added 72-h culture supernatants of ECV-NIL-1a-Flag on control cells. The supernatants of ECV-NIL-1a-Flag were not able to increase the motility of ECV-vectors (data not shown), suggesting that NIL-1aenhan- ces ECV304 cell motility by an intracellular effect. To exclude the possibility that the Flag tag could have any effect on cell motility by itself, we transfected ECV304 cells with pcDNA-NIL-1a in the absence of a Flag tag, and observed a significant increase of ECV-NIL-1a cell motility (3.29G

0.84 fold increase) as compared to control cells, in four experiments from two different transfections.

Next, we co-transfected cells with pcDNA-NIL-1a-Flag or empty pcDNA3 and pHook-icIL-1Ra1 or empty pHook and analyzed whether icIL-1Ra1 could antagonize the effect of NIL-1a propiece on cell motility. The increased cell motility induced by NIL-1a was completely antagonized by co-expression with icIL-1Ra1 (39G8 cells/field) (Fig. 3A).

The same results were obtained using transfected cells from three different double transfections. Fig. 3B illustrates

Fig. 3. Migratory response of NIL-1a-Flag and/or icIL-1Ra1 transfected ECV304 cells. The motility of empty vectors, NIL-1a-Flag, icIL-1Ra1, NIL-1a-Flag/icIL- 1Ra1 transfected ECV cells, was analyzed using transwell chambers (see Section2). (A) The migrating cells were counted in nine randomly chosen fields (150!) per well in triplicate wells. This figure is representative of six experiments, from three different double transfections. *p!0.05 vs. ECV-vectors;^yp!0.05 vs.

ECV-NIL-1a-Flag. (B) Staining of migrating cells with 0.5% crystal violet. (a) ECV-vectors, (b) ECV-NIL-1a-Flag, (c) ECV-icIL-1Ra1, (d) ECV-NIL-1a-Flag/

icIL-1Ra1 (magnification 150!). The number of migrating cells was increased in cells transfected with NIL-1a-Flag.

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results presented in Fig. 3A and shows photomicrographs of migrating cells on the lower side of the nucleopore filter stained with crystal violet. Together, these results suggest that preIL-1a enhances ECV304 cell motility by its NIL-1a propiece and this stimulatory effect is antagonized by icIL- 1Ra1.

3.4. icIL-1Ra1 antagonized the stimulatory effect of N terminal IL-1apropiece on the cell migration speed

Using a computerized system to analyze the speed of two- dimensional cell migration, we compared NIL-1a-Flag- and/

or icIL-1Ra1-transfected ECV304 cells with cells transfected with empty vectors. Overexpression of NIL-1a-Flag led to a fourfold increase of cell migration speed (23.91G3.67mm/h) as compared to ECV-vectors (5.65G2.28mm/h). Co- expression of icIL-1Ra1 with NIL-1a-Flag led to a significantly reduced average speed of migration (9.57G4.74mm/h) (Fig. 4).

3.5. Subcellular localization of icIL-1Ra1

By ELISA analysis, we examined the expression levels of icIL-1Ra1 in cytoplasmic and nuclear extracts of transfected ECV304 cells and of the A431 keratinocyte cell line, which constitutively expresses high amounts of icIL-1Ra1. icIL- 1Ra1 was predominantly localized in the cytosol of ECV- icIL-1Ra1, ECV-NIL-1a-Flag/icIL-1Ra1 and A431 cells (Table 1) but was also present to a lesser extent in the nucleus of these cells (!11%,Table 1). The results of ELISA on the subcellular localization of icIL-1Ra1 were confirmed by indi- rect immunofluorescence (data not shown).

4. Discussion

In the present study, we show that expression of preIL-1a increases the spontaneous motility of ECV304 cells in the absence of chemoattractants. It was reported that preIL-1apos- sess an IL-1 receptor binding affinity and biological activity very similar to that of mIL-1a[2] and that it could exert its activity as a soluble mediator[4]. The addition of exogenous soluble mIL-1a did not have any effect on the motility of ECV304 cells in our conditions, which suggest that the stimulatory effect of preIL-1awas independent of its interaction with cell surface IL-1 receptors. PreIL-1a has also been reported to exert intracellular functions, including the regulation of cell proliferation and senescence, apoptosis, migration, and cytokine production[5,6,8,9,20e22]via mechanisms that are independent of cell surface IL-1 receptors [5]. The 16 kDa NIL-1apropiece of preIL-1acontains a nuclear localization sequence and nuclear translocation of preIL-1a or NIL-1a propiece was shown to be required for these intracellular effects [5]. We show that expression of NIL-1a propiece also leads to a significant increase of the spontaneous motility as well as the cell migration speed of ECV304 cells. This effect was independent of its interaction with cell surface receptors and the release of other mediators external to the cells, a conclusion supported by the absence of this stimulatory effect when 72-h cell culture supernatants of ECV-NIL-1apropiece were incubated with control ECV304 cells. Our results demonstrate that the stimulatory effect of preIL-1aon cell motility is mediated intracellularly by its NIL-1apropiece.

We also show that the effect of preIL-1a and NIL-1a propiece on ECV304 cell motility was completely reversed by co-expression with icIL-1Ra1. The biological properties of sIL-1Ra as a natural inhibitor of extracellular IL-1 are well characterized[23]. It was reported that icIL-1Ra1 binds to IL-1 receptors with the same affinity as sIL-1Ra and inhibits the biological effects of IL-1 in vitro [13]. However, due to their intracellular localization, icIL-1Ra isoforms cannot inter- act with cell surface IL-1 receptors unless released under particular conditions. icIL-1Ra1 has been observed in the supernatants of epithelial cells after appropriate stimulation [24], and of HUVEC cells upon P2X7 receptor activation [25]. Consequently, one potential explanation of decreased preIL-1a responsiveness in ECV304 cells co-transfected

Fig. 4. Two-dimensional cell migration analysis. The migration distance of empty vectors, icIL-1Ra1, NIL-1a-Flag, NIL-1a-Flag/icIL-1Ra1 transfected ECV cells, was analyzed as described in Section 2. The migration of 20 cells/condition was measured and calculated as average speed of migration per hour (mm/h). *p!0.05 vs. ECV-vectors;^yp!0.05 vs. ECV-NIL-1a-Flag.

Table 1

Cytoplasmic and nuclear concentrations of icIL-1Ra1 Nuclear

extracts (ng/ml)

Cytoplasmic extracts (ng/ml)

% Nuclear vs.

cytoplasmic ECV-vectors Non-detectable Non-detectable

ECV-NIL-1a-Flag Non-detectable Non-detectable

ECV-icIL-1Ra1 0.28G0.088 13.6G5.4 3.1G1.7 ECV-NIL-1aFlag/icIL-1Ra1 0.97G0.4 21G2.5 5.1G2.5 A431 cells 23.59G2.62 229.35G9.47 10.3G0.8 IL-1Ra concentrations were measured by ELISA in the cytosolic and nuclear extracts of transfected ECV304 cells and A431 keratinocytes. Results show the meanGS.E.M. of triplicates of a representative experiment of five different experiments showing the same results.

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with icIL-1Ra1 in our studies could be the icIL-1Ra1 release into the supernatant, which would prevent the IL-1 binding to its receptor. Our observations argue against this possibility.

Measured supernatant levels of IL-1 and IL-1Ra in preIL-1a/

icIL-1Ra1 co-transfected cells were 0.26 ng and 3.2 ng/10⁶ cells, respectively. First, we observed that the addition of recombinant mIL-1a (up to 1 ng/ml) was devoid of effect on cell motility. Second, supernatant levels of IL-1Ra remained

!5 ng/ml in our cultures which is not sufficient to antagonize the IL-1 action. Several studies reported that much higher levels of IL-1Ra are required for a significant reduction of IL-1 action in transfected cells[15,16,26].

Several reports have proposed that icIL-1Ra1 may also exhibit unique functions inside cells. Ovarian cancer cells expressing high levels of icIL-1Ra1 displayed impaired IL-1- induced IL-8 and GRO mRNA expression likely due to the de- stabilization of the chemokine mRNA[14]. Also, icIL-1Ra1 expression in Caco-2 intestinal epithelial cells decreased IL- 1-stimulated IL-8 secretion[26]. This intracellular anti-inflammatory activity of icIL-1Ra1 was mediated through inhibition of p38 MAP kinase and NF-kB signal transduction pathways.

[15]. Recently, we showed that expression of preIL-1asignif- icantly decreased SaOS cell growth via an intracellular mode of action. Co-expression with icIL-1Ra1 did not reverse the growth inhibitory effect of preIL-1a[16]. Elevated production of preIL-1ahas also been implicated in growth inhibition of the transfected ECV304 cell line via an intracellular mechanism[5]. In agreement with this report, we observed a growth inhibition in ECV304 cells transfected with preIL-1a but co-transfection with icIL-1Ra1 did not reverse this growth inhibitory effect (personal observations), suggesting that intracellular effects of preIL-1a are not necessarily sensitive to inhibition by icIL-1Ra1. In contrast, we show in the present study that the effect of preIL-1a and NIL-1a on ECV304 cell motility is completely reversed by icIL-1Ra1 co-expression.

In addition, we observed that icIL-1Ra1 is present in the cytosol and the nucleus of transfected ECV304 cells. However, whether icIL-1Ra1 competes with NIL-1apropiece for nuclear translocation is not known.

ECV304 cells, originally described as a spontaneous trans- formant arising in a human umbilical vein endothelial cell (HUVEC) culture [27], have been used as a model cell line for endothelia over the last decade. These cells were recently described to be a variant of the T24 bladder carcinoma cell line that presumably contaminated the HUVEC culture in the laboratory where it was first isolated[28]. Although an identi- cal genotype has been established for ECV304 and T24 cells [28], it is clear from the published data that ECV304 cells ex- press some endothelial characteristics[29e34]. Recently, Suda et al.[35]studied the phenotype of ECV304 and T24 cells and showed that several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells.

Our findings demonstrate that preIL-1astimulates ECV304 cell motility via an intracellular mechanism and icIL-1Ra1 completely antagonizes this stimulatory effect. To our knowl- edge, this is the first report describing a relationship between

icIL-1Ra1 and preIL-1aregulating spontaneous cell motility, in vitro. This intracellular function of preIL-1a and icIL- 1Ra1, is novel. It remains, however, to be determined whether the ability of icIL-1Ra1 to antagonize the stimulatory effect of preIL-1aon cell motility extends to other cell types.

Acknowledgments

We wish to thank Franc¸oise Mezin, Lydia Bertrand and Christian Vesin for technical help. Special thanks are extended to Dana Hornfeld for editing help. This work was supported by a research grant from Roche Research Foundation and the Swiss National Science Foundation (grant 320000e107592 to C.G.).

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