In this study, a fibroblast growth-stimulating factor was purified from the exudate of car-rageenan-induced inflammation in rats.. We have purified and identified S100A9 as a new fibroblast gr
Trang 1Fibroblast growth-stimulating activity of S100A9 (MRP-14)
Futoshi Shibata, Katsuyoshi Miyama, Fumie Shinoda, Jun Mizumoto, Katsuhiko Takano
and Hideo Nakagawa
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan
Fibroblasts play a critical role in chronic inflammation
and wound healing In this study, a fibroblast
growth-stimulating factor was purified from the exudate of
car-rageenan-induced inflammation in rats The purified
protein was a disulfide-linked homodimer Amino acid
sequence analysis of the peptides generated by cleavage
with cyanogen bromide and proteinase V8 resulted in
identification of the protein as S100A9 Recombinant
S100A9 as well as its disulfide-linked homodimer
stimu-lated the proliferation of fibroblasts at a similar
con-centration of the purified protein The concon-centration of
S100A9 in the exudate was determined by immunoblot
analysis The total protein concentration in the exudate reached a maximum 4 days after carrageenan injection and then slightly decreased, whereas the concentration of S100A9 reached a maximum at day 3 and then decreased rapidly These studies show that S100A9 is present at a high concentration in the exudate of carrageenan-induced inflammation in rats, and that S100A9 stimulates pro-liferation of fibroblasts, suggesting that it plays a role in chronic inflammation
Keywords: carrageenan; fibroblast; growth; inflammation; S100A9
Granuloma is formed by a foreign body or infectious agents
and consists of epithelioid macrophages, multinucleated
giant cells and lymphocytes [1] Fibroblasts usually
sur-round granuloma, and play an important role in wound
healing These processes are mediated by growth factors and
cytokines, including platelet-derived growth factors [2],
transforming growth factor b [3], fibroblast growth factors
[4–6], and connective tissue growth factor [7] We have
purified and identified S100A9 as a new fibroblast
growth-stimulating factor (FGSF) from the exudate of
carrageenan-induced granulomatous inflammation in rats in this study
S100A9 [8], also known as calgranulin B [9] and MRP-14
[10,11], belongs to the S100 protein family and has two
Ca2+-binding EF-hand motifs S100A9 forms a
hetero-dimer with S100A8 [8], also known as calgranulin A [9] or
MRP-8 [10,11], and is expressed in granulocytes, monocytes
[11], and activated keratinocytes [12,13] Epithelioid cells in
foreign body granuloma also expressed S100A9 [14,15]
High serum concentrations of S100A9 are detected in cases
of cystic fibrosis [9], rheumatoid arthritis [11], systemic lupus erythematosus [16], Crohn’s disease [17], inflammatory bowel disease [18], and multiple sclerosis [19], and suggest
an important role of S100A9 in chronic inflammation It was reported that S100A9 bound zinc ions [20] and heparan sulfate glycosaminoglycans [21], also activated b2-integrin, Mac-1 on neutrophils [22] thereby controlling responsive-ness to neutrophil chemoattractants [23], as well as having macrophage-deactivating activity [14], antinociceptive activ-ity [24] and neutrophil chemotactic activactiv-ity [25] Calprotec-tin, a complexed form of S100A8 and S100A9, is known to inhibit microbial growth [26,27] and growth of fibroblasts
by chelating zinc ions [28] On the contrary, the present study provides evidence that S100A9 may function as a mitogen for fibroblasts in chronic inflammation
Materials and methods
Cells Mouse fetal fibroblasts, BALB/c 3T3 cells and normal rat kidney fibroblasts, NRK-49F cells were obtained from Japanese Cancer Research Resources Bank BALB/c 3T3 cells and NRK-49F cells were grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% (v/v) calf serum and 5% (v/v) fetal bovine serum, respectively
Proliferation assay For fibroblast growth-stimulating factors, proliferation of BALB/c 3T3 cells or NRK-49F cells was measured by the method described by Kueng et al [29] Cultured cells were placed into 96-well microtiter plates at a density of 1000 cells per well and allowed to grow for 24 h in the presence of
Correspondence to Futoshi Shibata, Department of Physiological
Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and
Pharmaceutical University, 2630 Sugitani, Toyama 930–0194, Japan.
Fax: + 81 76 4344656, Tel.: + 81 76 4347543,
E-mail: fshibata@ms.toyama-mpu.ac.jp
Abbreviations: ERK, extracellular signal regulated kinase; FGSF,
fibroblast growth-stimulating factor; GST, glutathione S-transferase;
MRP-14, myeloid-related protein-14; RAGE, receptor for advanced
glycation end products; XTT,
2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide.
Enzymes: BamHI (EC 3.1.21.4); glutathione S-transferase
(EC 2.5.1.18); horseradish peroxidase (EC 1.11.1.7); proteinase V8
(EC 3.4.21.19); SmaI (EC 3.1.21.4); thrombin (EC 3.4.21.5).
(Received 8 December 2003, revised 19 March 2004,
accepted 30 March 2004)
Trang 210% (v/v) calf serum Cells were then washed with
serum-free medium and incubated for 48–96 h in the medium
containing 2% (v/v) calf serum and sample solution Cell
number was measured by crystal violet staining
For S100A9, proliferation of NRK-49F cells was
meas-ured as described by Scudiero et al [30] NRK-49F cells
were inoculated at a density of 2000 cells per well into
96-well microtiter plates After 24 h, cells were washed with
serum-free medium and incubated for 48 h at 37C in the
medium containing 0.5% (v/v) fetal bovine serum and
experimental agents Prewarmed (60C) solution
contain-ing 50 lg of
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 0.38 lg of
phenazine methosulfate was added to each well After
incubation for 3 h at 37C, the plates were mixed and
absorbance at 450 nm was measured with a microplate
reader model 550 (Bio-Rad Laboratories)
Induction of air pouch-type inflammation
by carrageenan in rats
A 2% (w/v) solution of carrageenan (4 mL in saline,
Seakem 202, Marine Colloids Inc., NJ, USA) was injected
into preformed air pouches on the backs of male Wistar rats
(body mass: 170–200 g) [31] One, two, three, four, and
seven days after the injection, the rats were sacrificed by
cutting the carotid artery under light anesthesia and
granulation tissues and pouch fluid were then collected
Aliquots of the pouch fluid were frozen in liquid nitrogen
and stored at)80 C until use The concentration of protein
was determined using a Protein Assay Kit (Bio-Rad
Laboratories) The rats were treated in accordance with
procedures approved by the Animal Ethics Committee of
Toyama Medical and Pharmaceutical University
Purification of fibroblast growth-stimulating factors
All purification procedures except for RP-HPLC were
carried out at 4C The pouch fluid was collected on day 7
after carrageenan injection and centrifuged at 70 000 g for
60 min The resulting supernatant (day 7 exudate,
1000 mL) was adjusted to pH 4.5 with 9M HCl, and
stirred for 2 h After centrifugation at 13 000 g for 60 min,
the supernatant was brought to 38% saturation with
ammonium sulfate and stirred for 3 h, and then centrifuged
The resulting supernatant was precipitated by addition of
ammonium sulfate to 70% saturation This precipitate was
dissolved in 0.1Mphosphate buffer (pH 6.0), applied to a
CM-Cellulofine C-500 column (2.6· 47 cm; Seikagaku
Co., Tokyo, Japan) and eluted with 0.1M phosphate
buffer-150 mM NaCl (pH 6.0) Eluate was applied to a
heparin-Sepharose CL-6B column (1.6· 26 cm,
Amer-sham Biosciences, NJ, USA) and eluted with a linear
gradient from 0.1 to 2MNaCl in 50 mMTris/HCl buffer
(pH 7.0) Proteins which eluted between 0.6M and 1.2M
NaCl were pooled, concentrated, and chromatographed on
a Sephadex G-75 column (1.6· 94 cm; Amersham
Bio-sciences) equilibrated with phosphate-buffered saline
con-taining 0.01% (v/v) Brij-35 A peak fraction corresponding
to a molecular mass of about 20 kDa was pooled,
lyophilized, dissolved in 6M guanidine/HCl, and loaded
onto an ODS-120T column (0.46· 25 cm; Tosoh Co.,
Tokyo, Japan) at room temperature Proteins were eluted from the column by a linear gradient of acetonitrile from 0
to 50.4% (v/v) in 0.05% (v/v) trifluoroacetic acid at a flow rate of 0.8 mLÆmin)1 Finally, the major peak was rechro-matographed under the same conditions, and FGSF was isolated as a single peak
Amino acid sequence analysis of a fibroblast growth-stimulating factor
The purified FGSF was dissolved in 3Mguanidine hydro-chloride, 0.2M Tris/HCl (pH 8.2) at a concentration of
1 lgÆmL)1and reduced with 25% (v/v) 2-mercaptoethanol
at 40C for 3 h, and then carboxymethylated with 0.1M iodoacetic acid The carboxymethylated protein was puri-fied by HPLC on an ODS-120T column and fragmented by either cyanogen bromide cleavage or digestion with pro-teinase V8 from Staphylococcus aureus Resulting peptides were separated by RP-HPLC The N-terminal amino acid sequences of the peptides were determined by automated Edman degradation on an Applied Biosystems 470 A gas phase sequencer equipped with a 120 A on-line phenyl-thiohydantoin amino acid analyzer
Purification of recombinant S100A9 protein Total RNA from rat macrophages was reverse-transcribed S100A9 cDNA [32] was amplified from the cDNA by PCR, cloned between BamHI and SmaI sites of the glutathione S-transferase (GST) expression plasmid, pGEX4T2 (Amersham Biosciences), and sequenced Fusion protein expression was induced with 0.5 mM isopropyl thio-b-D -galactoside in Escherichia coli DH5a for 6 h at 28C After incubation, the bacteria was harvested by centrifugation and lysed by freezing and thawing, sonication, and addition
of 1% Triton X-100 The clear lysate was obtained by centrifugation and applied to a Glutathione Sepharose 4B column (Amersham Biosciences) GST-S100A9 fusion protein was eluted from the column with 10 mM glutathi-one)50 mM Tris/HCl (pH 8.0), chromatographed on a Sephadex G-50 column (Amersham Biosciences), equili-brated with phosphate-buffered saline to remove glutathi-one, and cleaved with thrombin (Amersham Biosciences) at
22C for 16 h This solution was passed through a Glutathione Sepharose 4B column to remove GST Finally, recombinant S100A9 was purified on an ODS-120T column (Tosoh Co.) using a linear gradient of acetonitrile from 28 to 40% (v/v) in 0.05% (v/v) trifluoroacetic acid, evaporated, dissolved in phosphate-buffered saline with 1 mMcalcium chloride and stored at – 20C Oxidation of the thiol group
of S100A9 was achieved with a copper–phenanthroline complex [33] The latter was removed by dialysis against
5 mMammonium acetate and the protein lyophilized The lyophilized sample was dissolved in phosphate-buffered saline
Production of polyclonal antiserum Polyclonal antiserum to S100A9 was raised in rabbits by subcutaneous injection of 1 mg of GST-S100A9 fusion protein emulsified in Freund’s complete adjuvant Two weeks after the primary injection, boosts of 0.5 mg of the
Trang 3fusion protein in Freund’s incomplete adjuvant were
injected every 2 weeks The rabbits were bled 2 weeks after
the final boost under anesthesia The rabbits were treated in
accordance with procedures approved by the Animal Ethics
Committee of Toyama Medical and Pharmaceutical
University
Gel electrophoresis
Exudates were diluted SDS buffer containing 2% (w/v) SDS
and 0.02% (w/v) bovine serum albumin SDS/PAGE was
carried out as described by Laemmli [34] using low molecular
mass markers and low-range rainbow molecular mass
mark-ers (Ammark-ersham Biosciences) as molecular mass standards
The gel was stained with Coomassie Brilliant Blue R 250
Immunoblotting
Proteins separated by SDS/PAGE were transferred onto
nitrocellulose membranes (Bio-Rad Laboratories) using
the Mini Trans-blot cell (Bio-Rad Laboratories) The
membranes were incubated with rabbit polyclonal anti-S100A9 serum and then with a horseradish peroxidase-conjugated goat anti-rabbit IgG (Caltag Laboratories, CA, USA) The reaction products were visualized with an ECL Western blotting detection system (Amersham Biosciences) and a luminoimage analyzer (LAS-1000 plus, Fuji Photo Film, Tokyo, Japan) Chemiluminescence was quantitated using the Science Laboratory 99 Image Gauge program (Fuji Photo Film) Chemiluminescence of the band linearly correlated with the amount of recombinant S100A9 (25 to
200 ng per lane) For quantitation of S100A9 in the exudates, three different amounts of recombinant S100A9 (80, 120 and 200 ng) were used in each assay as standards Statistical analysis
Data are expressed as mean ± SEM Student’s t-test was used for statistical analysis
Results
Purification of fibroblast growth-stimulating factors Fibroblast growth-stimulating factors were purified from the exudate of carrageenan-induced inflammation as des-cribed under Materials and methods and eluted from RP-HPLC as a major peak (peak 1) and a minor peak (peak 2) (Fig 1) The major peak was purified by rechromato-graphy on RP-HPLC We could not obtain an adequate amount of protein from peak 2 to continue analysis on it The purified FGSF (peak 1) gave a single band at 13.4 kDa under reducing condition and at 26 kDa under nonreducing condition, respectively (Fig 2) The N-terminal amino acid sequence of the purified FGSF could not be successfully performed, suggesting that the N-terminal amino acid is blocked Therefore, FGSF was carboxymethylated and treated with cyanogen bromide and proteinase V8; 4 (CN-1
to CN-4) and 12 (V-1 to V-12) peptides were then isolated
by RP-HPLC Although we could not determine any amino acid residues from peptides CN-4 and V-5, other peptides show a significant sequence similarity to rat S100A9 (Fig 3)
Fig 1 RP-HPLC separation of fibroblast growth-stimulating factors
from the inflammatoryexudate Proteins were eluted by a linear
gra-dient of acetonitrile from 0 to 50.4% (v/v) in 0.05% (v/v)
trifluoro-acetic acid Growth-stimulating activity of each fraction for BALB/c
3T3 cells was assayed at a concentration of 1 lgÆmL)1 Each column
represents the mean ± standard errors of six determinations.
Fig 2 SDS/PAGE analyses of a fibroblast growth-stimulating factor (FGSF) and recombinant S100A9 FGSF purified by rechromatography of peak 1 indicated in Fig 1, recombinant S100A9 and oxidized S100A9 (A9ox) were analyzed by SDS/PAGE in the absence (–) or presence (+) of 2-mercaptoethanol (final 10%) and stained with Coomassie brilliant blue.
Trang 4Production of recombinant S100A9
The coding region of cDNA for rat S100A9 was amplified
from macrophage RNA by RT-PCR The nucleotide
sequence of S100A9 cDNA was identical with that
regis-tered in GenBank/EMBL/DDBJ (T Imamichi; accession
number L18948) except for the replacement of G with C
that resulted in a change from arginine to serine at position
106 Nucleotide sequence data is available in the DDBJ/
EMBL/GenBank databases under the accession number
AB118215 Raftery et al [35] pointed out a cDNA
sequen-cing error due to the fact that mass spectrometry of S100A9
isolated from rat spleen found serine instead of arginine at
position 106
Recombinant S100A9 was produced using glutathione
S-transferase (GST) expression plasmid in Escherichia coli,
purified, and analyzed on SDS/PAGE (Fig 2) A single
band had a molecular mass of 13.6 kDa and was not altered
by reduction The sequence of N-terminal 10 residues of
recombinant S100A9 was identical to that of rat S100A9
(T Imamichi; accession number NP_446039) except for
two extra amino acids (Gly-Ser) at the N-terminus and the
lack of the initiator methionine After oxidation, most of
S100A9 existed as the disulfide-linked homodimer (Fig 2)
Growth-stimulating activity of FGSF and S100A9
As shown in Fig 4A, addition of FGSF purified from
exudate to the cultures of NRK-49F cells resulted in
dose-dependent stimulation of proliferation FGSF stimulated
proliferation of BALB/c 3T3 cells more efficiently (data not
shown) S100A9 and its disulfide-linked homodimer
stimu-lated proliferation of NRK-49F cells at concentrations
higher than 390 ngÆmL)1 (30 nM) and 260 ngÆmL)1
(10 nM), respectively (Fig 4B)
The concentration of S100A9 in exudate
Inflammation was induced by carrageenan on the back
of rats and granulation tissues and exudates were
collected (Fig 5) The volume of exudate continued to
increase even 7 days after carrageenan injection
How-ever, the wet weight of granulation tissue increased
rapidly until 4 days following the initial injection,
suggesting that granulation tissue formed 4 days after
carrageenan injection
To determine the concentration of S100A9 in the exudate, polyclonal antiserum was raised in rabbits against GST-S100A9 fusion protein The exudates collected (Fig 5) were diluted, electrophoresed and immunoblotted for S100A9 (Fig 6A) with three different amounts of recombinant
Fig 3 Comparison of the amino acid sequence
of rat S100A9 and peptides isolated from FGSF The identified amino acid residues of a cyanogen bromide-cleaved peptide (CN-3) and proteinase V8-digested peptides (V-2 to V-10) are aligned with the amino acid sequence of rat S100A9 (accession number NP_446039) Asterisks indicate amino acid residues identical with those of rat S100A9.
Fig 4 Growth-stimulating activityof FGSF and S100A9 NRK-49F cells were incubated with varying concentrations of FGSF purified from exudate (A) or S100A9 (B) in the presence of 1% (v/v) calf serum for 96 h (A) or 0.5% (v/v) fetal bovine serum for 48 h (B) Cell numbers were measured by crystal violet staining (A) or XTT staining (B) Each point represents the mean ± standard errors of six deter-minations Asterisks indicate significant differences (P < 0.01) from control.
Trang 5S100A9 as standards A band with a molecular mass of 13.6 kDa was detected under reducing condition A faint band of 60 kDa appears to be BSA added in SDS buffer, because the same band was detected without exudates The concentration of S100A9 was estimated by quantification of chemiluminescence of immunoblots of exudates and recom-binant S100A9 (Fig 6B) Protein concentration in the exudate reached a maximum 4 days after carrageenan injection and then slightly decreased, while the concentra-tion of S100A9 reached a maximum at day 3 and then decreased rapidly, indicating that the transient increase of S100A9 was specific, and not leakage from serum
Discussion
In the present study, a fibroblast growth-stimulating factor (FGSF) was purified from the exudate of carrageenan-induced inflammation in rats (Fig 1) Amino acid sequence analyses and SDS/PAGE indicated that the major protein
in FGSF was S100A9 homodimer (Figs 2 and 3) A relatively higher concentration (100 ngÆmL)1) of FGSF was required to stimulate proliferation of fibroblasts (Fig 4A), whereas fibroblast growth factor [36], epidermal growth factor [37], and connective tissue growth factor [38] were active at lower concentrations (0.4–3 ngÆmL)1) Recombin-ant S100A9 also stimulated the proliferation at a similar concentration to FGSF (Fig 4B), suggesting that the major active protein in FGSF was S100A9 Yui et al [28] reported that calprotectin, a complexed form of S100A8 and S100A9, inhibited the growth of human dermal fibroblasts presumably by the chelation of zinc ions This conflict may come from the difference of subunit composition Indeed, Newton and Hogg reported that S100A9 and S100A8/ S100A9 heterodimer showed a different biological activity [22]
S100B, another member of S100 family protein, stimu-lated proliferation of rat astroglial cells [39] and activated extracellular signal regulated kinase (ERK) in astrocytes [40] S100A12 and S100B bound a receptor for advanced glycation end products (RAGE) [41], which was reported to activate ERK [42] The receptor and signal transduction pathways leading to growth-stimulating activity of S100A9 have yet to be elucidated
S100A9 isolated from rat spleen was acetylated at the N-terminus after removal of the initiator methionine [35] Because we could not detect the N-terminal amino acid sequence of FGSF purified from the exudate, a similar modification may exist This N-terminal modification does not appear to affect growth-stimulating activity, as recombinant S100A9 also showed activity (Fig 4B) Although FGSF existed as a disulfide-linked homodimer (Fig 2), this oxidation may occur during purification steps However, both monomer and disulfide-linked homodimer forms of S100A9 stimulated the proliferation
of fibroblasts (Fig 4B)
The concentration of S100A9 in carrageenan-elicited exudates was very high (> 1 mgÆmL)1) during the forma-tion of granulaforma-tion tissue (Fig 6), and these high concen-trations of S100A9 were enough to stimulate fibroblast proliferation (Fig 4B) Our observations have led to the hypothesis that S100A9 contributes to the formation of granulation tissue by stimulating growth of fibroblasts
Fig 6 The concentration of S100A9 in the exudate of
carrageenan-induced inflammation in rats (A) Exudates (Fig 5) were analyzed by
immunoblotting for S100A9 in the presence of 2-mercaptoethanol
(final 10%) M: recombinant S100A9 (B) The concentration of
S100A9 was estimated by quantification of chemiluminescence
of immunoblots using a luminoimage analyzer Protein concentration
of the exudate was also determined Each point represents the
mean ± standard errors of 4–6 rats.
Fig 5 Formation of granulation tissue and retention of exudate.
Granulation tissue and exudate were collected on day 1–7 after
car-rageenan injection into a preformed air pouch on the back of rats.
Each point represents the mean ± standard errors of 4–6 rats.
Trang 6The valuable technical assistance of Mariko Kitahara, Kumi Ichinose,
Noriko Mannen and Manabu Kumakura is acknowledged.
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