neutrophil gelatinase-associated lipocalin) during mammalian embryonic development and in inflammation

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European Journal of Cell Biology 79, 165 ± 172 (2000, March) ´  Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb

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Expression of NRL/NGAL (neu-related lipocalin/ neutrophil gelatinase-associated lipocalin) during mammalian embryonic development and in inflammation Barbara Zeregaa, Silvia Cermellia, Beatrice Michelisb, Ranieri Canceddaa, c, Fiorella Descalzi Cancedda1)a, d a b c d

Istituto Nazionale per la Ricerca sul Cancro, Centro di Biotecnologie Avanzate, Genova/Italy Dipartimento di Scienze Motorie, Universita di Genova, Genova/Italy Dipartimento di Oncologia, Biologia e Genetica; Universita di Genova, Genova/Italy Centro di Studio per la Neurofisiologia Cerebrale, Consiglio Nazionale delle Ricerche; Genova/Italy

Received July 10, 1999 Received in revised version November 9, 1999 Accepted November 12, 1999

Lipocalin ± cartilage ± muscle ± development ± inflammation The neu-related lipocalin (NRL) is a protein overexpressed in rat mammary cancer induced by activated neu (HER-2/cerbB2). This protein belongs to the family of the lipocalins or low molecular weight proteins able to bind and transport small hydrophobic molecules. The NRL homologue in mouse is SIP24, an acute phase protein induced in the animal by turpentine injection; the human homologous protein is NGAL expressed in granulocytes and epithelial cells in pathological conditions, such as inflammation and malignancy. We have investigated NRL expression in developing rat embryos. By immunolocalization we have shown localization of the protein in the hypertrophic region of growth plate cartilage. NRL was particularly enriched in prehypertrophic chondrocytes. In addition, we observed localization of the protein in forming skeletal muscle fibres and in the myocardium of developing heart. In agreement with the immunolocalization data, by in situ hybridization we have demonstrated the presence of the specific mRNA in the same tissues. At an early stage of differentiation, cultured rat embryo-derived chondrocytes did not express NRL; nevertheless expression of the protein was induced in these cells by treatment with an inflammatory agent, such as LPS. By Western blot analysis with specific antibodies we showed protein synthesis by cultured myoblasts also in the absence of LPS treatment, but only when forming myotubes were observed in culture. Stimulation of myoblast cultures with LPS resulted in an enhancement of the NRL expression in well formed myotubes. Our data suggest a role of

Dr Fiorella Descalzi Cancedda, Centro di Biotecnologie Avanzate, Largo Rosanna Benzi, 10, I-16132 Genova/Italy, e-mail: [email protected], Fax: ‡ 39 010 5737405.

1)

NRL in cartilage and muscle differentiation. NRL expression was induced by inflammatory agents. We wish to propose that the expression of NRL in hypertrophic chondrocytes and forming myotubes is part of a ªphysiologicalº acute phase response occurring during cartilage and muscle development. In this manuscript we also report that NRL is not detectable by immunolocalization in adult cartilage (both articular and tracheal) from normal subjects. On the contrary articular cartilage from osteoarthritic patients was highly positive for the presence of NRL/NGAL. Interestingly the expression of this protein is also activated during neoplastic transformation of chondrogenic lineage cells.

Introduction Lipocalins are a family of more than 50 known proteins, mainly extracellular, involved in the binding, transport and presentation of small lipophilic molecules. These proteins share a common folding pattern resulting in an eight-stranded antiparallel b barrel. The cup-shaped barrel encloses the hydrophobic substrate-binding pocket. The specific ligands for some lipocalins have been identified. These ligands include retinol (Berman et al., 1987; Newcomer et al., 1984), retinoic acid (Newcomer, 1993), biliverdin (Holden et al., 1987; Huber et al., 1987), odorants (Bianchet et al., 1996; Bocskei et al., 1992; Tegoni et al.,1996), progesterone (Balbin et al., 1990; Pearlman et al., 1973), and fatty acids (Descalzi Cancedda et al., 1995). The function of these proteins is different depending on the ligand and for some of them is unknown. Some lipocalins play an important role in cell regulation: serum retinol-binding protein is responsible for distributing the mitogenic and differentiating retinoid vitamin A from the liver to peripheral target cells (Sivaprasadarao and Findlay, 1988), protein HC has a mitogenic effect on both T and B cells

0171-9335/00/79/03-165 $12.00/0

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166 B. Zerega, S. Cermelli et al.

and inhibits stimulation of lymphocytes by antigens (Akerstrom and Logdberg, 1990). NRL (neu-related lipocalin) is a rat lipocalin overexpressed in tumors initiated by the activated neu (HER2/c-erbB-2) oncogene, extremely potent in inducing mammary cancer (Stoesz and Gould, 1995). The mouse homologue of NRL is SIP24 (Flower et al., 1991), a lipocalin produced by quiescent Balb/c 3T3 cells and inducible by many factors, including serum, FGF-2, prostaglandin F2a, phorbol ester and dexamethasone (Davis et al., 1991; Hamilton et al., 1985). Sip 24 is also inducible by LPS in macrophages (Meheus et al., 1993) and is an acute phase protein expressed in liver during the acute phase response (APR) induced by turpentine in mouse (Liu and Nilsen-Hamilton, 1995). The human homologue of NRL is NGAL (neutrophil gelatinase-associated lipocalin), a lipocalin stored in specific granules of the human neutrophil (Kjeldsen et al., 1994). NGAL is constitutively expressed during a narrow window of maturation of the granulocyte precursors in the bone marrow, i. e. the myelocytes/metamyelocytes (Borregaard et al., 1995). The ligand for NGAL is unknown, but binding of the bacterial formyl peptide fMLP has been demonstrated (Sengelùv et al., 1994) suggesting that NGAL might be a scavenger of bacterial products at sites of inflammation. NGAL synthesis has been shown also in epithelial cells as a consequence of inflammation and malignancy (Nielsen et al., 1996). From these data, a role of the homologous lipocalins NRL, NGAL and SIP24 in pathological conditions and in inflammation can be hypothesized. We have isolated and characterized in our laboratory a lipocalin specifically binding fatty acids (Ex-FABP) (Descalzi Cancedda et al., 1995) and developmentally regulated in chick embryo. Ex-FABP is expressed during chondrogenesis and bone formation (Descalzi Cancedda et al., 1988; Dozin et al., 1992; Manduca et al., 1989), myogenesis and heart development (Gentili et al., 1998) and is also present in high concentrations in granulocytes (Dozin et al., 1992). More recently we have observed that Ex-FABP is also expressed in inflammation and modulated by inflammatory and anti-inflammatory agents (Cermelli et al., 2000). The aim of the present study was to demonstrate a NRL involvement during tissue differentiation in embryonic development with the hope to find a possible homologue of Ex-FABP in mammals. In fact, any previous attempt to identify a homologous mammalian protein by antibody cross-reaction or by Ex-FABP cDNA probe crosshybridization has failed. We pointed at NRL/NGAL/SIP24 among other lipocalins (all sharing about 25 ± 30% homology with Ex-FABP) because of the expression of NGAL in granulocytes and the association of SIP24 with inflammation. Here we show NRL expression ªin vivoº in rat embryos. By immunohistochemistry and in situ hybridization, NRL was localized in the hypertrophic region of developing cartilage, in the newly forming myofibers and in the myocardium of the developing heart. The protein was not expressed in detectable amounts in cultures of proliferating chondrocytes derived from embryonic tibiae at an early stage of differentiation, but its expression was induced in the same cells by treatment with the inflammatory endotoxin LPS. In cultures of rat myoblasts undergoing maturation to myofibers NRL release into the culture medium was observed by Western blot analysis. By immunohistochemistry NRL was localized in well formed fibers. Treatment of cultured myoblasts with LPS resulted in a strong enhancement of NRL expression. In normal adult cartilage (articular and tracheal) NRL was not expressed. In

articular cartilage, NRL/NGAL was expressed in pathological conditions such as osteoarthritis, confirming the association of NRL/NGAL with inflammatory conditions. In addition NRL/ NGAL was highly expressed in chondrosarcoma, a cartilaginous tumor of bone. In summary, our data indicate that NRL is developmentally regulated in cartilage and muscle differentiation and that, in these tissues, its expression is modulated by inflammatory agents. The same protein is expressed in the pathological status of adult cartilage. A possible common role in differentiation and in inflammation is suggested.

Materials and methods Cell culture

Cultured myoblasts were obtained from limbs of 17-days-old rat embryos, as previously described (Stoesz and Gould, 1995). Briefly, isolated tissues were digested in 0.05% trypsin (Gibco BRL Inc., Grand Island, NY) in phosphate-buffered saline (PBS) at 37 8C for 10 ± 20 min. After proteolytic digestion, tissues were fragmented by repeated pipetting, debris were removed by filtration through a sterile nylon gauze, and cells were collected by centrifugation. The cell suspension was first plated in standard tissue culture dishes for 30 min, in order to reduce the number of contaminating fibroblasts, more rapidly adhering to the plastic, and then replated on collagen-coated dishes. The culture was grown in Dulbeccos minimum essential medium supplemented with 15% horse serum and 5% chick embryo extract. Chondrocytes were obtained from 17-days-old rat embryo tibiae according to published procedures (Descalzi Cancedda et al., 1988). Cells were obtained from cartilaginous bone by proteolytic digestion at 37 8C and plated at confluence in order to maintain the cartilaginous phenotype. Cells were cultured in F12 culture medium supplemented with 10% fetal calf serum. Ascorbic acid was added after two days at a concentration of 0.1 mg/ml. Cells were maintained in culture for 5 additional days before treatment; medium was changed every 2 days. When indicated, for both chondrocytes and myoblasts, the endotoxin LPS from E. coli (Sigma Chemical Co. St Louis, MO) was added at a concentration of 10 mg/ml in serum-free medium.

Cell culture labeling, polyacrylamide gel electrophoresis and immunoprecipitation

Culture dishes were washed with PBS, incubated at 37 8C for 2 hours with methionine-free medium and labeled for 2 hours with [35S]methionine (100 mCi/ml) as previously described (Descalzi Cancedda et al., 1988). Labeling of chondrocytes was always performed in the presence of ascorbic acid (100 mg/ml). Aliquots of culture media were run for protein analysis on SDSPAGE as described (Descalzi Cancedda et al., 1988), except otherwise indicated, in reducing conditions. Polyacrylamide gel concentration was 15%. Immunoprecipitation of specific proteins was performed as previously described (Gentili et al., 1998).

Western blot analysis of secreted proteins

Myoblasts were cultured for 9 days and were stimulated with LPS (10 mg/ml) overnigth in serum-free medium. Chondrocytes were cultured for 7 days and were stimulated with LPS in serum-free medium for 5 additional days changing the medium every two days. Media from control and stimulated cells were collected and concentrated. Concentrated conditioned culture media were loaded on a 15% SDSpolyacrylamide gel electrophoresis. Electrophoresis was performed in reducing condition. After electrophoresis the gel was blotted to a BA85 nitrocellulose membrane (Schleicher and Schuell GmbH, Dassel, Germany) according to the procedure described by Towbin et al. (Towbin et al., 1979). The blot was saturated for 16 hours with 3% nonfat cow milk in TTBS buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20), washed several times with TTBS and incubated with rabbit anti-NRL IgG antibodies for 3 hours at room temperature. After

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washing, the detection was performed by an AP-conjugated anti-rabbit IgG (Boehringer Mannheim GmbH, Mannheim, Germany) using BCIP/NBT from ICN Biomedicals (Costa Mesa CA) as a substrate. Recombinant NRL protein produced in a baculovirus system and rabbit anti-NRL IgG antibodies were kindly supplied by Dr. M. N. Gould (Stoesz and Gould, 1995).

Immunohistochemistry

For immunohistochemical localization of NRL/NGAL protein in the tissues, biopsies were embedded in paraffin. Serial sections (5 mm) were made, dewaxed and treated with methanol/ H2O2 (49 : 1) for 30 minutes to inhibit endogenous peroxidases. Sections were then treated with 1 mg/ml hyaluronidase in PBS for 20 minutes at 37 8C and washed with PBS. After incubation with goat serum for 20 minutes to reduce nonspecific binding of the secondary antibody, the specific antibody was added for 1 hour at room temperature. Sections were washed several times with PBS and challenged with biotinylated goat anti-rabbit IgG (Jackson Laboratory Inc., West Grove, PA USA) and peroxidaseconjugated egg-white avidin (Jackson Laboratory, Inc.). After additional washing of the sections with PBS and 50 mM Na acetate, pH 5, the peroxidase activity was visualized by enzymatic modification of the 3-amino-9-ethylcarbazole substratum (3-amino-9-ethylcarbazole 0.4% in dimethylformamide : 50 mM Na acetate, pH 5 : 30% H2O2; 100 : 900 : 1) during 15 minute incubation in the dark at room temperature. Sections were counterstained with Harris hematoxylin and mounted with Gel/mount from Biomeda Corp. (Foster City, CA/ USA). Slides were observed and photographed with a Zeiss Axiophot. Antibodies: Antibodies directed against recombinant rat NRL and against human NGAL were kindly supplied by Dr. M. N. Gould.

In situ hybridization

Rat embryos (17 days) were decalcified with EDTA, pH 8.0, for 7 h at 37 8C. Sections (5 mm) of paraffin-embedded rat embryos were dewaxed and treated sequentially with: a) 0.2 N HCl for 15 min; b) 20 mg/ml proteinase K in 50 mM Tris-HCl, pH 7.6, 5 mM EDTA for 7 min, at room temperature; c) 0.4% glycine in PBS; d) 4% paraformaldehyde in PBS for 15 min; e) 1/400 acetic anhydride in 0.2 M triethanolamineHCl, pH 8.0, for 10 min. Hybridization of tissue sections was performed in a hybridization mixture containing: 10% dextran sulfate, 3  SSC, 0.1 mg/ml salmon sperm DNA, 0.125 mg/ml yeast tRNA, 10 mg/ml poly(A), 1 mg/ml sodium pyrophosphate in 1 M Tris, pH 7.4, 50% formamide. For probe preparation we used a 601-bp rat NRL cDNA cloned into pCRII plasmid, kindly supplied by Dr. M. N. Gould. Probe, labeled with digoxigenin-UTP (DIG RNA Labeling Kit ± SP6/T7; SIGMA), was synthesized using T7 and SP6 RNA polymerase. Probe was used at a concentration of 0.75 ng/ml hybridization mix. After 2 h prehybridization (without the probe) at room temperature, slides were incubated overnight at 50 8C with hybridization mix. After hybridization, slides were washed at first in 50% formamide, 2  SSC at 50 8C for 30 min, then twice in 2  SSC at 54 8C. Nonhybridized transcript was digested with 10 mg/ml RNase A in STE (4  SSC, 20 mM Tris-HCl, pH 7.6, 1 mM EDTA) for 30 min at 37 8C. Sections were washed three times at 54 8C for 15 min with: 1) 2  SSC, 2) 1  SSC, 3) 0.2  SSC. Dehydrated sections were air dried. Slides were washed with buffer A (1.5 M NaCl; 0.1 M Tris-HCl, pH 7.5; 2 mM MgCl2; 0.3% Tween 20) for 30 min at room temperature. The transcript was detected using antidigoxigenin antibody conjugated with alkaline phosphatase (Boehringer Mannheim, Germany) 1 : 400 in buffer A for 2 h at 37 8C. Slides were washed twice in buffer A and twice in buffer C (0.1 M NaCl; 0.1 M TrisHCl, pH 9.5; 5 mM MgCl2). Then sections were incubated in colordevelopment solution, 0.3 mg/ml nitroblue tetrazolium, 0.15 mg/ml 5bromo-4-chloro-3-indolyl phosphate, 2 mM levamisole. Color development was stopped by immersion of the slides into buffer C. Sections were weakly counterstained with nuclear fast red, and mounted with DPX mountant.

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Results NRL expression in rat embryos

Localization of NRL protein in sections of 17-days-old rat embryo was performed by immunohistochemistry with specific polyclonal antibodies raised against the recombinant protein (Fig. 1). In embryonic tibia sections, NRL protein was observed in the growth plate, in the region of the prehypertrophic chondrocytes (Fig. 1A). At higher magnification it can be seen that the protein is localized in the cytoplasm of the prehypertrophic/hypertrophic chondrocytes and partly diffused in the surrounding extracellular matrix (Fig. 1B). On the contrary, most of the cells in the proliferating chondrocyte region were not stained by the specific antibodies. Other characteristic localizations of the protein in the 17days-old rat embryo were in the forming skeletal muscle fibers (Fig. 1C) and in the developing myocardium (Fig. 1D). In situ hybridization experiments with the probe specific for the NRL mRNA, confirmed the transcription of the gene by the prehypertrophic/hypertrophic chondrocytes in the growth plate (Fig. 1E, F) and by fusing myoblasts (Fig. 1G, H).

NRL expression in myoblast and in chondrocyte cultures: LPS induces and enhances the NRL synthesis

Cultured rat myoblasts undergo myogenic differentiation in vitro. Proliferating myoblasts become postmitotic, fuse to form multinucleated myotubes and express a number of musclespecific proteins, including the muscle-specific myosins. Cultures of rat myoblasts at late developmental stage, when well formed myotubes were clearly detectable, were stimulated with the bacterial toxin LPS. Western blot analysis of conditioned culture media from both control and stimulated cells was performed (Fig. 2A). In agreement with the observed expression by fusing myoblasts in vivo, NRL was detectable in medium from control cells (lane 2). Interestingly, supplement of the culture with the inflammatory agent LPS resulted in a strong enhancement of the NRL secreted by the cells (lane 3). Quantitation of NRL produced by the cells, by scanning of the Western blot, revealed that LPS-treated cells released into the culture medium about 2 mg/ml of the protein (3 cm culture dish). We should note that a lower electrophoretic mobility of the NRL secreted by cultured myoblasts and chondrocytes with respect to the recombinant protein mobility has been always observed. This finding can be explained with a difference in posttranslational modification of the native protein with respect to the recombinant protein. Metabolic labeling of the cell culture with [35S]methionine, followed by immunoprecipitation with NRL-specific antibodies, confirmed the synthesis of NRL in LPS-stimulated myoblasts and its release into the culture medium (Fig. 3). Double immunofluorescence staining of the cultures was also performed. In LPS-stimulated cultures NRL colocalized with sarcomeric myosin in formed muscle fibers (Fig. 4A, C); in unstimulated cells the low amount of NRL expressed was still colocalized with sarcomeric myosin in formed muscle fibers (Fig. 4B, D). Chondrocyte cultures were obtained from the whole tibia of 17-days-old rat embryos. At this embryonic stage the tibia is completely cartilaginous, although in the diaphysis region some chondrocytes already show a hypertrophic phenotype (Fig. 1A, B). When cultured in adhesion, these chondrocytes grow mainly as proliferating chondrocytes, and only a small

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Fig. 1. Localization of the NRL protein in the developing rat embryo. Immunolocalization of NRL was performed on sections of tibia (A, B), forming muscles (C) and developing heart (D) of 17-days-old rat embryos. B) is an enlargement of the boxed area in A). Localization of the NRL-specific mRNA in the 17-days-old developing rat embryo. In

situ hybridization was performed with antisense (E, G) and sense (F, H) DIG-riboprobe. Hyperthrophic chondrocytes in a long bone (E) and a muscle from the same embryo (G) show specific staining (arrowheads). Bars: A: 160; B, C: 40; D: 640 mm; E ± H: 40 mm.

number of more differentiated cells is sometimes present in the culture. This chondrocyte population did not release NRL at detectable level into the culture medium. Nevertheless, when the cells were stimulated with LPS, a band corresponding to NRL was clearly identified by Western blot analysis in the conditioned medium (Fig. 2B). Similar results were obtained also by stimulation of the cells with IL-6 (not shown).

Expression of NRL/NGAL in human osteoarthritis

Samples from osteoarthritic patients were obtained in hip replacement surgery; controls were obtained from two young and healthy deceased subjects. Immunolocalization of the protein was performed with antibodies directed against the rat NRL and the human NGAL. Identical results were obtained

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Fig. 2. Western blot analysis of media conditioned by cultured myoblasts and chondrocytes. Conditioned medium was run on a 15% SDS polyacrylamide gel in reducing conditions, blotted to a nitrocellulose filter and subjected to immunoblot analysis using NRL polyclonal antibodies. A: Lane 1) 0.25 mg of recombinant NRL protein was loaded as positive control; lane 2) control myoblasts; lane 3) myoblasts treated with LPS. B: Lane 1) control chondrocytes; lane 2) chondrocytes treated with LPS.

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Fig. 3. Proteins released by cultured myoblasts treated with the inflammatory bacterial endotoxin LPS. Rat myoblasts cultured for 12 days were labeled with [35S]methionine. Labeled proteins released in the media were analysed by 15% SDS-PAGE in reducing condition. Lane 1) control myoblasts; lane 2) myoblasts treated with LPS for 16 hours before collection of the culture medium; lanes 3, 4) immunoprecipitation by specific NRL polyclonal antibodies of labeled proteins released in the culture medium in the absence (3) and in the presence of LPS (4). Numbers on the left refer to molecular weight.

with both antibodies. Expression of the protein was observed in the pathological articular cartilages but not in control normal cartilages (Fig. 5A). On the whole, the data we have obtained point to the NRL/NGAL as a protein expressed in a specific cartilage status, i. e. inflammation or degeneration of adult articular cartilage and differentiation of embryonic growth plate cartilage. The NRL protein was never detected in the tracheal cartilage of adult rat, a permanent cartilage that does not undergo differentiation or inflammatory degeneration (Fig. 5B).

Expression of NRL/NGAL in human chondrosarcoma

Fig. 4. Coexpression of NRL and heavy-chain myosin in myoblasts cultured for 14 days. A, C) myoblasts treated with LPS for 16 hours, B, D) control myoblast. A, B) anti NRL; C, D) anti MF-20 (anti-myosin heavy chain). Double immunofluorescence was performed as described in Materials and methods. The expression of NRL is observed when myotubes are formed, at the same time as myosin is synthesized, and is enhanced by LPS treatment. Bar: 70 mm.

The expression of NRL/NGAL can be activated also during neoplastic transformation. Interestingly, when a biopsy from a human chondrosarcoma (classified as low malignancy grade) was analysed, a strong positive staining was observed in virtually all the cells (Fig. 5C). A portion of the biopsy was minced with a surgical blade, digested with hyaluronidase and a protein extract prepared. This protein extract was analysed by Western blot with antibodies against NRL (Fig. 6) or against NGAL (not shown). The same result was obtained with both antibodies. A single band with an electrophoretic mobility slightly lower than the mobility of recombinant NRL was identified (Fig. 6, lanes 1 ± 2).

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Fig. 5. A) Immunolocalization of NRL/NGAL in human osteoarthritic cartilage. Biopsies from the hip joint articular cartilage of osteoarthritic patients were decalcified and embedded in paraffin prior to sectioning. a) Osteoarthritic cartilage from a 54-years-old woman; b) control cartilage from a normal 16-years-old woman. Sections were stained with anti-NRL polyclonal antibodies. Bar: 5 mm. B) Immunolocalization of NGAL in adult trachea. Rat trachea was isolated and

embedded in paraffin. Longitudinal paraffin sections were stained with antibodies directed against NRL. b) Higher magnification of boxed area in a). Bar: a 40 mm; b 10 mm. C) Immunolocalization of NGAL in a human chondrosarcoma. A biopsy from a chondrosarcoma from a 57years-old woman was embedded in paraffin. Sections were obtained and stained with antibodies against NGAL. a) chondrosarcoma b) negative control. Bar: 5 mm.

Discussion

inflammatory agents repressed the synthesis of the protein physiologically expressed and induced by LPS (Cermelli et al., 2000). Based on this and on other observations we have proposed that Ex-FABP represents a stress protein physiologically expressed in chick tissues where active remodelling is taking place during development and in tissues characterized by an acute phase response due to pathological conditions.

We have previously shown that the synthesis of Ex-FABP, a 21 kDa secreted fatty acid-binding lipocalin expressed in hypertrophic cartilage, muscle and heart during chick embryonic development and in blood granulocytes, was increased by the inflammatory agents LPS and interleukin 6, whereas anti-

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Fig. 6. Western blot analysis of a chondrosarcoma-derived protein extract. A portion of the biopsy was minced with a surgical blade and digested with hyaluronidase (1 mg/ml in PBS, 90 min at 37 8C). After the digestion, the cell suspension was sonicated and precipitated with TCA. The TCA-precipitated material was loaded on a 15% SDSPAGE. Western blot analysis was performed with antibodies against NRL. Lane 1) chondrosarcoma protein extract; lane 2) control recombinant NRL.

The purpose of this study was to identify a possible homologue of Ex-FABP in mammals. We focused on the NRL/NGAL/Sip24 since it was reported in the literature that, as the Ex-FABP, this lipocalin is expressed in human granulocytes (NGAL; Borregaard et al., 1995; Kjeldsen et al., 1994) and is associated with inflammation both in mice (Sip24) (Liu and Nilsen-Hamilton, 1995; Meheus et al., 1993) and in humans (NGAL) (Nielsen et al., 1996). In this report we have shown the expression of this lipocalin during rat embryo development and we have investigated its possible association with pathological conditions, including neoplasia. We have observed that, as the Ex-FABP in chick embryos, NRL in rat embryos is localized in the prehypertrophic/hypertrophic cartilage region of the bone growth plate, in forming skeletal muscle myofibers and in developing myocardium. NRL/ NGAL was also localized in human osteoarthritic articular cartilage and in chondrosarcoma, a cartilaginous tumor of bone. In addition, similarly to the finding that Ex-FABP is induced by inflammatory agents in cultures of chick chondrocytes and myoblasts, we observed that in the cultures of rat myoblasts stimulated with LPS, the expression of NRL was highly enhanced and that in cultures of rat chondrocytes expression of NRL was induced by LPS. On the overall, these data highly strengthen our original hypothesis that the mammalian homologue of the chick ExFABP is the NRL/NGAL/Sip24 protein. Unfortunately, we were unable to detect a binding between the recombinant NRL and long chain unsaturated fatty acids. We should note that the native protein in our systems always shows a slower electrophoretic mobility with respect to the available recombinant NRL. This is shown also for NRL protein from rat mammary carcinomas of differing etiologies (Stoesz and Gould, 1995). The NRL protein present in neu tumors was of varying molecular weights, nearly all larger than the recombinant protein suggesting substantial posttranslational processing. The same difference was also observed for NGAL from tumor samples of breast carcinomas with respect to the recombinant NGAL (Stoesz et al., 1998). It is possible that posttranslational modifications occurring in the native protein are important for the correct folding of the protein and for the binding of its ligand. In dyschondroplastic chickens a high amount of Ex-FABP was observed in the lesion area. In osteoarthritic chickens ExFABP was expressed in affected articular cartilage (Cermelli et al., 2 000). Both pathologies are characterized by chondrocyte

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degeneration not necessarily associated with an inflammatory status. As in the case of the chick Ex-FABP, the expression of the NRL/NGAL appears to be enhanced during an inflammatory response. Inflammation is accompanied by cell degeneration and death. We suggest a correlation between the lipocalin expression and cell survival and we would like to speculate that these molecules have also an anti-inflammatory role. It has been reported that the bacterial formyl peptide fMLP binds to NGAL suggesting that NGAL might be a scavenger of bacterial products at sites of inflammation (Sengelùv et al., 1994). This finding is in agreement with an anti-inflammatory role of NGAL/NRL, but the physiological ligand during acute phase response induced by turpentine in mouse (Liu and Nielsen-Hamilton, 1995) or in pathological conditions like inflammation and malignancy (Nielsen et al., 1996; Stoesz and Gould, 1995; Stoesz et al., 1998) or in physiological stress condition like differentiation of chondrocytes and myoblasts is not known. A potential function for the lipocalins may be suggested by their putative ligand, i. e. long chain fatty acids. Inflammation is characterized by an increased metabolism of arachidonic acid. An increase in the fatty acids present in the cell microenvironment may be due also to an increased cell death and degradation of cell components observed in inflammation. Ex-FABP and NRL/NGAL released by the cells in the extracellular microenvironment may act as scavenger for fatty acids and protect the cells against the toxic effect of these molecules. It may be not fortuitous that the growth plate hypertrophic cartilage is characterized by a high number of degenerating cells both in chick, where Ex-FABP is expressed, and in rat, where NRL is expressed. The synthesis of a high amount of NGAL by chondrosarcoma cells is worth a specific comment. Gene expression of ngal was observed, although at widely variable levels, also in primary breast cancers (Stoesz et al., 1998). In this case the expression levels were higher than the level in normal breast parenchyma only in a subset of tumors. Obviously we are not in the position to make any prediction with respect to NRL expression in chondrosarcomas from different patients. Nevertheless it is to mention that tumor cells can be envisaged as continuously proliferating cells in a microenvironment otherwise hostile that favors cell degeneration and death. Alternatively, the synthesis of an extracellular fatty acidbinding protein might be required for the transport and metabolism of the fatty acids released by the cells, due to an increased turnover of membrane phospholipids, and/or of fatty acids and lipid-derived metabolites acting as local hormones. A major reorganization of cell membranes, is observed both in inflammation (activation of macrophages, aggregation of platelets, altered vessel permeability) and during differentiation and morphogenesis processes (further differentiation and degeneration of hypertrophic chondrocytes, myogenesis). The role of arachidonic acid and its metabolites as local hormones is well known (Jurivich et al., 1994; Vane et al., 1994). The finding that in chicken, Ex-FABP as well as specific metalloproteases, transferrin and chemotactic activity are expressed both in the embryonic growth plate hypertrophic cartilage and in inflammation of different tissues, supports the idea that characteristic APR responses are activated during endochondral bone formation and possibly during other cell differentiation pathways. In particular, in the case of endochondral bone formation, the expression of these molecules by hypertrophic chondrocytes does not require a specific inflammatory stimulus and it is instead the result of a differentiation

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program followed by the cells. In this study we have reported that NRL/NGAL is localized in the growth plate hypertrophic cartilage region and forming myofibers of rat embryos and in the human osteoarthritic articular cartilage. In addition we have found that the synthesis of the protein by cultured chondrocytes and myoblasts is highly enhanced by the inflammatory agent LPS. We suggest that, as in the case of chick embryogenesis, responses considered characteristic of an acute phase response and a local inflammatory status are ªphysiologicallyº activated during endochondral bone formation, muscle development and possibly other cell differentiation pathways also in mammalian embryogenesis. Acknowledgements. This work was partially supported by funds from Associazione Italiana per la Ricerca sul Cancro and MURST. We thank Prof. Michael N. Gould, Department of Human Oncology, University of Wisconsin, Madison, WI, USA for making available to us antibodies against NRL and NGAL, NRL cDNA probe and recombinant NRL protein. We thank Dr. Maria Teresa Costantini, Banca dellosso ± Dipartimento di Scienze Motorie, Universita di Genova and Prof. Rodolfo Capanna, Istituto di Chirurgia Oncologica e Ricostruttiva, CTO, Firenze for the generous supply of biopsies of normal and pathological cartilages. We also thank Ms. Barbara Minuto for editing the manuscript.

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