Tài liệu Báo cáo khoa học: FGF-2, IL-1b and TGF-b regulate fibroblast expression of S100A8 doc

FGF-2, IL-1b and TGF-b regulate fibroblast expression
of S100A8
Farid Rahimi, Kenneth Hsu, Yasumi Endoh and Carolyn L. Geczy
Inflammatory Diseases Research Unit, School of Medical Sciences, University of New South Wales, Sydney, Australia

Keywords
FGF-2; fibroblasts; interleukin-1b; S100A8
gene; TGF-b
Correspondence
C. Geczy, Inflammatory Diseases Research
Unit, School of Medical Sciences, The
University of New South Wales, Sydney,
NSW 2052, Australia
Fax: + 61 293851389
Tel: + 61 293851599
E-mail:
Website: />(Received 3 March 2005, revised 28 March
2005, accepted 5 April 2005)
doi:10.1111/j.1742-4658.2005.04703.x

Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-b (TGF-b) regulate fibroblast function, differentiation and
proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+binding proteins and are now accepted as markers of inflammation. They are
expressed by keratinocytes and inflammatory cells in human ⁄ murine wounds
and by appropriately activated macrophages, endothelial cells, epithelial cells
and keratinocytes in vitro. In this study, regulation and expression of S100A8
and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon c
(IFNc), tumour-necrosis factor (TNF) and TGF-b did not induce the
S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h. The FGF-2 response was strongly enhanced and prolonged
by heparin. Interleukin-1b (IL-1b) alone, or in synergy with FGF-2 ⁄ heparin
strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not
induced under any condition. Induction of S100A8 in the absence of

S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by
FGF-2 and IL-1b was partially dependent on the mitogen-activated-proteinkinase pathway and dependent on new protein synthesis. FGF-2-responsive
elements were distinct from the IL-1b-responsive elements in the S100A8
gene promoter. FGF-2- ⁄ heparin-induced, but not IL-1b-induced responses
were significantly suppressed by TGF-b, possibly mediated by decreased
mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblastlike cells 2 and 4 days post injury; numbers declined by 7 days. Up-regulation
of S100A8 by FGF-2 ⁄ IL-1b, down-regulation by TGF-b, and its timedependent expression in wound fibroblasts suggest a role in fibroblast
differentiation at sites of inflammation and repair.

Fibroblasts are heterogeneous stromal resident cells
that participate in wound healing, fibrosis ⁄ scarring
and immune ⁄ inflammatory processes [1,2] by contributing to leukocyte recruitment ⁄ accumulation, angiogenesis, matrix metabolism, and protection against

oxidative damage [3,4]. Numerous factors including extracellular matrix (ECM) components, some
cytokines, prostaglandins, reactive oxygen species
(ROS), and growth factors [5] modulate fibroblast
function.

Abbreviations
ActD, actinomycin D; BCS, bovine calf serum; BM, bone marrow; BMF, bone-marrow-derived fibroblast-like cells; C ⁄ EBP, CCAAT ⁄ enhancer
binding protein; CM, culture medium; DAPI, 4,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; DPBS, Dulbecco’s
phosphate-buffered saline; DTT, dithiothreitol; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum;
FGF, fibroblast growth factor; Hepes, N-2-hydroxyethylpiperazine-N ¢-2-ethanesulfonic acid; HPRT, hypoxanthine phosphoribosyl-transferase;
HRP, horseradish-peroxidase; IFNc, interferon c; IL-1b, interleukin-1b; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein; LPS,
endotoxin; mS100A8, murine S100A8; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; mOxS100A8, HOCl-oxidized murine
S100A8; PKC, protein kinase C; ROS, reactive oxygen species; SPF, splenic ‘primary’ fibroblast-like cells; TGF-b, transforming growth
factor-b; TNF, tumor-necrosis factor.

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Expression and regulation of S100A8 in fibroblasts

S100 Ca2+-binding proteins may have evolved from
a calmodulin ancestor [6] and are implicated in numerous intra ⁄ extracellular processes and can act as Ca2+
sensors [7]. Some (e.g. S100A6 (calcyclin) [8], S100A4
[9] and S100A11 [10,11]) have been implicated in fibroblast growth and differentiation. S100A12 acts as
a pro-inflammatory ‘cytokine’ [12,13] and S100A12
mRNA is induced by interleukin-1a (IL-1a) and tumor
necrosis factor (TNF) in bovine corneal fibroblasts
[14]. S100B is also expressed by fibroblasts [15] and
may be involved in regulation of growth arrest and
apoptosis [16], stimulation of cell proliferation [17]
and protection against apoptosis [18]. Other S100
proteins also have proliferative and anti-apoptotic
effects [8,9,19].
S100A8 and S100A9 (calgranulins A and B; MRP8
and MRP14) have been associated with leukocyte differentiation, inflammation and wound healing [20,21].
They are proposed to be involved in reorganization of
the keratin cytoskeleton and differentiation of keratinocytes and in antibacterial or antioxidant defense in
the wounded or normal epidermis [20–22]. Some functions are dependent on S100A8–S100A9 heterodimers,
e.g. arachidonic acid binding, antimicrobial defense
and regulation of NADPH-oxidase activity (reviewed
in [20]). S100A9 and the S100A82–S100A9 complex
(calprotectin) stimulate IL-8 production by airway epithelial cells, hence potentially amplifying neutrophilic
inflammation in chronic pulmonary disease [23].
Murine S100A8 is a potent leukocyte chemoattractant
[24] and intracellular S100A8 and S100A9 essentially

regulate phagocyte migration by integrating the calcium and mitogen activated protein kinase (MAPK)
transduction signals thereby controlling reorganization
of the phagocyte microtubular system [25]. Expression
of S100A8 in the absence of S100A9 in activated murine macrophages [26,27] and keratinocytes [22], and
presence of S100A8 without S100A9 in the low density
lipoprotein (LDL) proteome [28] is strong evidence
that S100A8 does not depend on S100A9 for structural
stability [29] and strengthens the proposal for independent function. We showed that S100A8 scavenges
O2– and hypochlorite, suggesting a role in oxidative
defense [22,30,31]. The S100A8 gene is up-regulated by
anti-inflammatory mediators [27], corticosteroids [32]
and by oxidative stress [22] indicating a protective
function. Other S100 proteins are also implicated in
cellular responses to oxidative stress. In particular in
keratinocytes, S100A2 oxidation and translocation
were proposed as early markers of oxidative stress and
were markedly attenuated in malignant keratinocytes,
favoring a role in oxidant defense rather than in tumor
proliferation [33].
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F. Rahimi et al.

Here we show that factors important in wound healing regulate S100A8, but not S100A9, in fibroblasts.
Fibroblast growth factor-2 (FGF-2) and IL-1b
strongly induced S100A8 via a MAPK-dependent
pathway. Responses to FGF-2 were amplified by heparin and there was strong synergy between FGF-2 and
IL1b. The protein was cytoplasmic. TGF-b suppressed
S100A8 induction by FGF-2 but not by IL-1b, suggesting important regulatory differences, and promoter
analysis confirmed different enhancer elements regulating induction by IL-1b and FGF-2. In a rat incisional

wound, immunohistochemical studies showed S100A8
expression in fibroblast- and macrophage-like cells,
keratinocytes and neutrophils in the incision area. We
propose that S100A8 may be involved in pathways
regulating fibroblast growth and differentiation, possibly by regulating intracellular redox, at sites of
inflammation and ⁄ or repair ⁄ remodeling.

Results
FGF-2 induces S100A8 mRNA in 3T3 fibroblasts
Initially, induction of murine S100A8 (mS100A8)
mRNA in 3T3 fibroblasts was variable, suggesting
that, like microvascular endothelial cells [34], cell–cell
contact may be important. Fibroblast monolayers
grown to various states of confluence, were stimulated
with FGF-2 ± heparin and harvested after 24 h.
Unstimulated confluent (C, Fig. 1) or subconfluent
(not shown) 3T3 cells had little detectable S100A8
mRNA whereas the bone-marrow (BM) RNA was
positive (BM, Fig. 1). FGF-2 (1.5 nm) weakly induced
the S100A8 gene and responses gradually increased
from  21–51% of maximum in cells grown from
 30–80% confluence; levels were maximal at confluence (Fig. 1). Heparin modulates FGF-2-receptor binding and activity [35] and although it had no direct
influence (Fig. 6), S100A8 mRNA increased approximately two-fold in confluent 3T3 cells stimulated with
FGF-2 and heparin compared to FGF-2 alone. Potentiation was minimal in cells grown to  80% confluence (Fig. 1) indicating that cell–cell contact is
important for optimal induction of S100A8 mRNA
expression in 3T3 fibroblasts. Real-time RT-PCR confirmed that S100A9 was not induced in confluent cells
stimulated with FGF-2 with ⁄ without heparin.
Kinetics of induction of S100A8 mRNA
in 3T3 fibroblasts
mS100A8 mRNA induction in FGF-2-heparin-activated fibroblasts was dose- and time-dependent. Up to

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F. Rahimi et al.

Expression and regulation of S100A8 in fibroblasts

A

Fig. 1. S100A8 mRNA induction in fibroblasts depends on confluence. 3T3 cells grown to approximately 30, 80 and 100% confluence were stimulated with FGF-2 (1.5 nM) in the presence (+) or
absence (–) of heparin (1 ImL)1) for 24 h. Total RNA was examined by Northern blotting using an S100A8 riboprobe and a rat 18S
rRNA oligoprobe as indicated. BM indicates murine bone-marrow
RNA (positive control) and C, the negative control (unstimulated
cells). The graph indicates percentage maximum of the normalized
response. Similar results observed in at least three experiments.

0.15 nm FGF-2 with 1 ImL)1 heparin did not induce
detectable mRNA (Fig. 2A), whereas 1.5 nm FGF-2
and heparin induced strong responses that were maximal with 3 nm FGF-2 (Fig. 2A); 6 and 15 nm FGF-2
induced mS100A8 mRNA levels that were  80 and
60% of maximal expression, respectively (Fig. 2A),
suggesting production of a suppressor. Heparin generated maximal responses at 1 and 10 ImL)1 (Fig. 2B)
whereas higher amounts (50 and 100 ImL)1) reduced
responses to  72 and 48% of maximum, respectively,
in cells costimulated with 1.5 nm FGF-2 (Fig. 2B)
possibly due to soluble heparin-mediated inhibition of
FGF-2-receptor binding [35]. For subsequent experiments, 1.5 nm FGF-2 with 1 ImL)1 heparin were
used to stimulate confluent fibroblasts.
S100A8 mRNA induction in 3T3 fibroblasts activated with FGF-2 was evident after 8 h and in the presence of heparin, mRNA levels increased in parallel up
to 12 h when the response to FGF-2 was maximal and

gradually declined over 36 h (Fig. 2C). Potentiation by
heparin was most apparent at 18 h, when mRNA levels were approximately double those with FGF-2 alone
at 12 h. In FGF-2-heparin-stimulated cells, S100A8
mRNA levels were maintained for up to 36 h and
declined to 20% of maximum by 48 h (Fig. 2C).
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B

C

Fig. 2. Dose- and time-dependence of induction of S100A8 mRNA
by FGF-2 and heparin in confluent 3T3 cells. (A) Confluent 3T3 cells
treated with FGF-2 (1.5 pM-15 nM) and heparin (1 ImL)1) for 24 h
and total RNA analyzed by Northern blotting; percentage maximum
response is given in the bar graph; C and BM indicate unstimulated
fibroblasts’ and bone-marrow RNA, respectively. (B) 3T3 cells treated with 1.5 nM FGF-2 were costimulated with increasing amounts
of heparin (0.01–100 ImL)1). (C) Kinetics of induction of S100A8
mRNA. 3T3 cells treated with FGF-2 (1.5 nM) or FGF-2 ⁄ heparin
(1 ImL)1) were harvested at the times indicated and Northern
analysis performed. Similar results observed in three different
experiments.

Because the mS100A8 gene in elicited macrophages
and other cells [26,34] is induced by particular proinflammatory mediators, endotoxin (LPS, 250–
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Expression and regulation of S100A8 in fibroblasts

1000 ngỈmL)1), interferon c (IFNc, 500 mL)1), or
TNF (30 ngỈmL)1) were tested. No S100A8 mRNA
was detected in 3T3 cells stimulated for 24 h although
mRNA was substantially augmented with a combination of LPS, IFNc, and TNF and confluence influenced levels (not shown). Induction was maximal after
24 h and decreased to  47% of maximum by 48 h
(not shown).
IL-1b-stimulated 3T3 fibroblasts express S100A8
mRNA
IL-1b is a strong inducer of the S100A8 gene in microvascular endothelial cells [34]. IL-1b induced S100A8
mRNA in confluent 3T3 fibroblasts in a dose- and
time-dependent manner. As little as 1 mL)1 was
effective and responses were maximal with 10–
20 mL)1 (Fig. 3A); 10 mL)1 IL-1b were used routinely. mRNA was detected after 8 h, gradually
increased to 28% of maximum after 24 h, was maximal at 32 h and declined to  30% of maximum after
48–52 h (Fig. 3B).
S100A8 mRNA levels in 3T3 cells stimulated with
FGF-2 ⁄ heparin almost tripled when costimulated with
0.1 mL)1 IL-1b and were augmented  7.5-fold
with 2 mL)1. Maximal up-regulation was with

A

F. Rahimi et al.

Table 1. S100A8 mRNA levels assayed in activated 3T3 cells by
real-time RT-PCR. 3T3 cells unstimulated or treated with IL-1b
(10 mL)1), FGF-2 (1.5 nM) + heparin (1 ImL)1) or their combination for 24 h. mS100A8, mS100A9, and HPRT mRNA levels were
quantitated by real-time RT-PCR. S100A8 expression was normalized to that of HPRT and expressed as fold-increase compared to
medium control. The data are means and standard errors of duplicate measurements. S100A9 mRNA was not detected.
Treatment

S100A8 induction

S100A9 induction

Medium control
IL-1b
FGF-2 + heparin
FGF-2 + heparin + IL-1b

1.0
97.0
74.54
5907

0
0
0
0

±
±
±
±

0.064
8.48
13.89
390.2

10 mL)1 IL-1b which generated a 14.2-fold increase
compared to 10 or 20 mL)1 IL-1b alone, or FGF-2
and heparin without IL-1b (Fig. 3C). To quantitate
this more accurately, relative S100A8 mRNA levels
were analyzed by real-time RT-PCR. Table 1 shows
that IL-1b (10 or 20 mL)1) induced mRNA levels
similar to those induced by FGF-2 and heparin. The
magnitude of synergy was more apparent, with  580fold more S100A8 mRNA in cells costimulated with
FGF-2, heparin and IL-1b compared to those stimulated with FGF-2 and heparin.

B

C

Fig. 3. Effects of IL-1b on S100A8 mRNA
induction. (A) Northern analysis of mRNA
from confluent 3T3 cells stimulated with the
given doses of IL-1b for 24 h. Results represent three experiments. (B) 3T3 cells stimulated with 10 mL)1 IL-1b were harvested
at the times indicated. The line graph indicates normalized levels of S100A8 mRNA.
Similar results observed in two experiments. (C) Northern blot analysis of confluent 3T3 cells stimulated for 24 h with FGF-2
(F, 1.5 nM) and heparin (H, 1 ImL)1) in the
presence of increasing doses of IL-1b; data
representative of at least three different
experiments. (D) Confluent SPF stimulated
with FGF-2 and heparin and IL-1b at the
doses indicated. The bar graph indicates
normalized levels of S100A8 mRNA.

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D


F. Rahimi et al.

Induction of S100A8 mRNA in primary murine
fibroblast-like cells
Because fibroblasts are phenotypically and functionally
heterogeneous and possess unique subpopulations even
in the same tissue [36,37] and because 3T3 fibroblasts
may not be representative of tissue fibroblasts, the
S100A8 gene regulation was assessed in different fibroblast populations and RNA from splenic ‘primary’
fibroblast-like cells (SPF) and bone marrow-derived
fibroblast-like cells (BMF) were analyzed. Confluent
SPF contained no detectable S100A8 mRNA by
northern analysis (Fig. 3D) and cells stimulated for 24 h
with IL-1b (samples 8 and 9), FGF-2 (sample 10), or
FGF-2 and heparin (samples 4 and 5) did not express
the gene. IL-1b and FGF-2 combined (samples 6 and 7,
Fig. 3D) weakly induced mRNA ( 38–47% of maximum), responses at 24 h were maximal with 6 nm FGF-2,
1 ImL)1 heparin and 10 mL)1 IL-1b (sample 3)
and half-maximal with 2 mL)1 IL-1b and 1.5 nm
FGF-2 (sample 2, Fig. 3D). The same pattern was
observed with BMF stimulated with FGF-2, heparin
and IL-1b (Fig. 6D). RT-PCR confirmed that S100A9
was not induced in primary murine fibroblast-like cells
by FGF-2, heparin and IL-1b stimulants (not shown).

Expression and regulation of S100A8 in fibroblasts

Promoter analysis in 3T3 fibroblasts
To examine mechanisms of transcriptional regulation
of the S100A8 gene by IL-1b, and FGF-2 plus heparin, 5¢-flanking sequences upstream of the transcription initiation site, untranslated intron 1 and sequences
upstream of exon 1, were used to evaluate activities of
deletion constructs after transient transfection into 3T3
cells (Fig. 4). Marked differences between FGF-2heparin- and IL-1b-induced responses were seen with all
constructs. IL-1b did not change the luciferase activity
of any construct although northern blotting of mRNA
preparations of IL-1b-stimulated cells in the same
experiment was positive (not shown). Levels of luciferase activity after FGF-2 ⁄ heparin stimulation were
similar for all constructs, with two- to fourfold increases over luciferase activity in unactivated cells. The
region )94 to )34 bp contained the essential promoter
because its deletion completely abrogated luciferase
activity. The region )178 to )94 bp is responsible for
luciferase activity in unactivated cells (basal activity)
because deletion strongly reduced basal activity but
elements involved in FGF-2 enhancement were
retained. Consensus motifs for a number of transcription factors, including CCAAT ⁄ enhancer binding

Fig. 4. Luciferase activity of S100A8 promoter deletion constructs in fibroblasts stimulated with FGF-2 + heparin or IL-1b. Various S100A8
5¢-truncated mutant constructs shown on the left were used and the transcription initiation site was at position +1. Promoterless is the parent vector (pGL2) and Promoter is the promoterless with an SV40 promoter (pGL2-promoter). 3T3 cells were transiently transfected with various constructs and a Renilla luciferase construct. The cotransfected cells were left unstimulated (in CM) or treated with FGF-2 (1.5 nM) +
heparin (1 ImL)1) or IL-1b (10 mL)1) for 48 h. The firefly luciferase activity, normalized to that of Renilla luciferase, was compared with
the normalized activity of unstimulated promoter-transfected cells and expressed as normalized fold-induction. The right bars represent averaged luciferase activities obtained from duplicates from at least three separate experiments. Error bars represent standard deviation of the
mean.

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Expression and regulation of S100A8 in fibroblasts

F. Rahimi et al.

protein (C ⁄ EBP), Ets, and E box are located within
this region. Constructs not containing the 1st exon and
intron ()178–0 bp) generated positive, but relatively
weak luciferase activities but in the same proportions,
indicating that this region still contains elements essential for gene induction by FGF-2.

A

The MAPK pathway is involved in S100A8 mRNA
induction by FGF-2 and IL-1b in 3T3 fibroblasts

B

The MAPK pathway is implicated in S100A8 mRNA
induction by IFNc and LPS in macrophages [26]. In
3T3 cells, PD 098059 (MAPK kinase (MEK) inhibitor,
50 lm) suppressed FGF-2- and FGF-2-heparininduced S100A8 mRNA by  65 and 62%, respectively, and SB 202190 [c-Jun N-terminal kinase
(JNK) ⁄ p38 inhibitor, 10 lm] by  50% (Fig. 5A).
Similarly, PD 098059 (50 or 75 lm) or SB 202190 (10
or 20 lm) reduced responses to IL-1b by  70–83%
(Fig. 5B) indicating converging pathways in FGF-2- ⁄
heparin- and IL-1b-induced S100A8 mRNA induction
in fibroblasts.
Cycloheximide completely abrogated the S100A8

gene in 3T3 cells stimulated with FGF-2 ± heparin
(Fig. 5C) or IL-1b (Fig. 5D), indicating a requirement
for de novo protein synthesis.

C

D

TGF-b suppresses FGF-2-induced mS100A8 mRNA
in 3T3 and primary fibroblasts
Because TGF-b modulates fibroblast function and phenotype [38,39], its effect on S100A8 gene expression
was tested. 3T3 cells cultured with TGF-b, heparin,
or a combination of both, did not express S100A8
mRNA (Fig. 6A). S100A8 mRNA induction by
FGF-2 ± heparin was almost completely abrogated in
cells simultaneously cultured with TGF-b for 24 h
(Fig. 6A). Quantitative RT-PCR showed 34.3-fold
induction of S100A8 mRNA by FGF-2 ⁄ heparin which
decreased approximately sixfold with TGF-b; mRNA
levels induced by FGF-2 alone were halved in cells coincubated with TGF-b (Fig. 6B). No S100A9 mRNA
was found in any of the samples tested by RT-PCR in
this experiment (not shown).
In marked contrast to its effect on the FGF-2- ⁄ heparin-induced responses (lane 10, Fig. 6C), TGF-b
(8 pm-0.8 nm) did not reduce S100A8 mRNA levels
induced by IL-1b (lanes 2–4, Fig. 6C). However, the
high mRNA levels with a combination of IL-1b, FGF2 and heparin (lane 5, Fig. 6C) were reduced only by
 74% with 0.08 nm TGF-b (lane 7, Fig. 6C); concentrations up to 0.8 nm did not increase suppression
(lane 8, Fig. 6C). S100A8 mRNA induced with
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Fig. 5. Involvement of the MAPK pathways and de-novo protein
synthesis in induction of S100A8 mRNA in fibroblasts. (A) 3T3 cells
stimulated (24 h) with FGF-2 (F, 1.5 nM) or FGF-2 and heparin
(H, 1 ImL)1) with ⁄ without 4-h preincubation with PD 098059
(PD, 50 lM) or SB 202190 (SB, 10 lM) as indicated. (B) 3T3 cells stimulated (24 h) with IL-1b (10 mL)1) with ⁄ without 4-h preincubation with PD 098059 (50–75 lM) and SB 202190 (10–20 lM). Data
represent three experiments. (C) 3T3 fibroblasts stimulated with
FGF-2 (F, 1.5 nM) and heparin (H, 1 ImL)1) for 24 h with or without
4-h preincubation with 5 lgỈmL)1 cycloheximide (CHX) as indicated.
C, mRNA from unstimulated cells; BM, murine bone-marrow RNA.
(D) 3T3 cells stimulated with IL-1b (10 mL)1) for 24 h with or without 4-h preincubation with cycloheximide (5 and 10 lgỈmL)1) as indicated. Similar results observed in four experiments.

FGF-2 ⁄ heparin and IL-1b in BMF decreased by
 67% with 0.08 nm TGF-b (Fig. 6D).
To clarify the mechanism of TGF-b suppression,
3T3 cells incubated for 20 h with FGF-2 ⁄ heparin in
the presence ⁄ absence of TGF-b were treated with
actinomycin D (ActD) for another 20 h to inhibit further transcription. Cells stimulated with FGF-2 or
FGF-2 ⁄ heparin harvested 20 h after treatment with
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F. Rahimi et al.

A

Expression and regulation of S100A8 in fibroblasts

A

B

B

C

Fig. 7. Expression of S100A8 protein in 3T3 cells. (A) 20–25 · 106
confluent 3T3 cells unstimulated (S1, L1) or stimulated with 1.5 nM
FGF-2 and 1 ImL)1 heparin with (S2, L2) or without 10 mL)1
IL-1b (S3, L3) for  30 h. Total cell-associated (L) and secreted (S)
protein was immunoprecipitated, SDS ⁄ PAGE was in the presence
of DTT (100 mM), and western analysis using anti-mOxA8 IgG.
Recombinant murine S100A8 (4, 40 and 100 ng) was the positive
control. Results obtained in two experiments. (B) Approximately
20–25 · 106 cells were either unstimulated (lane 1) or stimulated
(lane 2) for  30 h; cell lysates and supernatants (600 lL) were subjected to immunoaffinity purification through an anti-mS100A8 affinity column and C4 RP-HPLC. The collected fractions were then
analyzed by SDS ⁄ PAGE and western blotting in the absence of
DTT. Recombinant mS100A8 (25 ng) was used as the positive
control.

D

FGF-2 ⁄ heparin and treated with ActD were reduced
by  49 and 66%, respectively, indicating reduced
mRNA stability. Similar results were obtained in two
experiments (not shown).
S100A8 protein in activated 3T3 fibroblasts
Fig. 6. TGF-b regulates S100A8 mRNA induction by FGF-2. (A) Northern analysis of mRNA from 3T3 cells stimulated with FGF-2
(F, 1.5 nM), heparin (H, 1 ImL)1), TGF-b (T, 0.08 nM) alone or in the
combinations indicated for 24 h; C, unstimulated cells. (B) RT-PCR
analysis of the RNA samples used in (A). Levels of S100A8 mRNA
were compared to those of HPRT in the corresponding samples and

ratios of S100A8 mRNA ⁄ HPRT are indicated for each sample. (C)
Cells stimulated with FGF-2 (F, 1.5 nM) ⁄ heparin (H, 1 ImL)1) with
or without IL-1b (10 mL)1) in the presence or absence of increasing concentrations of TGF-b (8 pM-800 pM) as indicated. BM, total
bone-marrow RNA. (D) Confluent BMF treated for 24 h with the indicated doses of FGF-2, heparin, IL-1b and TGF-b.

ActD had 126% of the mRNA levels of untreated
cells, indicating somewhat increased mRNA stability ⁄ accumulation. In the presence of TGF-b, mRNA
levels from cells stimulated with FGF-2 or
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S100A8 was not detected in unprocessed lysates or
supernatants of 2.5–3 · 106 stimulated ⁄ unstimulated
confluent 3T3 cells by western blot analyses or by double-sandwich ELISA (detection limit ¼ 0.03 nm [40]).
Pull-down experiments, to concentrate the protein
from a larger number of cells (20–25 · 106), showed
no S100A8 in supernatants or lysates of unstimulated
cells (S1 and L1, respectively, Fig. 7A). Low levels
were present in supernatants (S2, Fig. 7A) but S100A8
was mainly cell-associated in cells stimulated for 30 h
with FGF-2 ⁄ heparin and IL-1b (L2, Fig. 7A). Separation was initially performed in the presence of dithiothreitol (DTT) to increase the yield by reducing
disulfide-linked complexes and the characteristic monomeric mass of 10 kDa (rA8; Fig. 7A) was confirmed.
In an alternative approach, supernatants and lysates
of activated cells were concentrated using an affinity
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Expression and regulation of S100A8 in fibroblasts

support and bound protein subjected to C4 reversephase HPLC. Recombinant mS100A8 elutes as a single
peak at 19.8–20 min (not shown). Because no major

peak were obtained (possibly due to low levels),
fractions were collected between 17.25 and 21.25 min
(covering the expected retention-time range for native
mS100A8 monomer and dimer), and lyophilized. No
mS100A8 was detected in fractions from supernatants
(not shown) or lysates (lane 1, Fig. 7B) of unstimulated cells. Western blotting of the three fractions from
lysates of stimulated cells collected over 18.25–20.25 min
(lane 2, fraction collected at 18.25–19.25 min, Fig. 7B)
contained components of molecular mass 20 kDa, with
the same migration profile as dimeric mS100A8,
contained in the positive control (lane 3, Fig. 7B). No
mS100A8 monomer (10 kDa) was detected. This was
unexpected as the same conditions have yielded monomeric and complexed forms of mS100A8 in other
stimulated cell types [34,41].

F. Rahimi et al.

No S100A8 was found in unstimulated 3T3 cells
stained with preimmune IgG or an antibody against
HOCl-oxidized mS100A8 (mOxS100A8) (Fig. 8A,B,
respectively); 4,6-diamidino-2-phenylindole (DAPI)stained nuclei were evident. Similarly, stimulated cells
stained with the nonimmune IgG were unreactive
(Fig. 8C). Approximately 10–30% of 3T3 cells stimulated with FGF-2 ⁄ heparin ⁄ IL-1b showed bright cytoplasmic fluorescence (Fig. 8D). When costimulated
with TGF-b, S100A8-positive cells dropped to 5% of
total (Fig. 8E). Confocal microscopy clearly showed
localization of S100A8 (red fluorescence) in the cytoplasm with no obvious associations with cytoskeletal
structures (Fig. 8F).
S100A8 expression in rat dermal wounds
To assess in vivo expression of S100A8 in fibroblasts,
rat dermal wound tissue was used. Examination of

Fig. 8. Immunofluorescent detection of
S100A8. Confluent 3T3 cells grown in slide
chambers were unstimulated (A, B) or
stimulated for 30 h with 1.5 nM FGF-2,
1 ImL)1 heparin and 10 mL)1 IL-1b in
the absence (C, D, F) or presence of
0.08 nM TGF-b (E). Permeabilized fixed cells
stained with nonimmune rabbit IgG (A, C) or
anti-mOxA8 (B, D, E, F) followed by antirabbit IgG-Alexa-Fluor-568 and the nuclear
stain, DAPI, were analyzed by fluorescent ⁄ confocal microscopy. A high-power
image of a representative S100A8-positive
fibroblast stimulated with FGF-2, heparin
and IL-1b is shown (F). Similar results were
obtained in two experiments. Magnifications: (A–E) 400·; (F) 600·.

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F. Rahimi et al.

wounded rat skin 2 days post injury showed an intensely anti-S100A8-immunoreactive scab, containing
S100A8-positive neutrophils (Fig. 9Aa), impinging on
the injured, and surrounding the normal epidermis. In
normal epidermis, the superficial more differentiated
keratinocytes reacted with anti-mS100A8 more intensely than those in the stratum basale which contains
the proliferating keratinocytes. In the dermis, small
dilated microvessels containing intensely stained

Expression and regulation of S100A8 in fibroblasts

S100A8-positive neutrophils were evident, representing
the in situ positive controls (Fig. 9Ab). Based on
morphology, extravascular S100A8-positive cells were
identified as macrophages or extravasating neutrophils. Some spindle-shaped fibroblast-like cells closely
apposed to, and aligned with collagen fibers were also
relatively intensely mS100A8-positive (Fig. 9Ab)
although staining was heterogeneous. These were identified as fibroblast-like cells, based on morphology,

Fig. 9. Immunohistochemical localization of S100A8 in rat dermal wounds. (A) Immunostaining of rat dermal wound 2 days after injury. (Aa)
Low-power (200 ·) view of the wounded dermis beneath the neutrophil-rich S100A8-positive scab (S). Black arrows indicate S100A8-positive
fibroblast-like cells apposed to collagen fibers indicated by C. Neovessels and sebaceous glands are indicated by V and SG, respectively.
Delineated inset in (Aa) corresponds to (Ab) (400 ·) where neutrophils and macrophage-like cells are indicated by red and blue arrows,
respectively. Black arrows point to S100A8-positive fibroblast-like cells. (B) Staining of rat wound 4 days after injury. Anti-S100A8 IgG
(Ba, Bb) and nonimmune IgG (Bc) were used. (Ba) The scab (S) is evident along the wounded epidermis and encroaching normal uninjured
keratinocytes (K). Some hair follicles (H), small vessels (V) and sebaceous glands (SG) surrounded by collagen (C) fibers are evident. (Bb) An
area of granulation tissue showing macrophage-like (blue arrows) and fibroblast-like cells (black arrows) around neovessels (V) were S100A8positive. Some S100A8-negative fibroblast-like cells are indicated by asterisks. (Bc) Immunostaining of the 4-day wound with nonimmune
IgG. (C) Anti-mS100A8 immunostaining of rat wound 7 days after injury. (Ca) Low-power view of an area of mature granulation tissue and
scar formation rich in collagen fibers and fibroblasts stained with anti-mS100A8 IgG. The epidermal keratinocytes are evident to the right of
the wounded area (K). The inset shown in (Ca) corresponds to (Cb) where S100A8-negative fibroblast-like cells are indicated by arrows.

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Expression and regulation of S100A8 in fibroblasts

location in the granulation tissue and alignment with
collagen fibers as no appropriate specific marker to
detect rodent fibroblasts was available. Similarly, in
a mouse model of delayed-type hypersensitivity
responses, about 10% of fibroblast-like cells were
S100A8-positive (S. Leong and C.L. Geczy; unpublished data).
In 4-day wounds, granulation tissue (macrophages,
fibroblasts, neovessels and collagen) was well-established and most spindle-shaped fibroblast-like cells and
macrophages stained positively with anti-mS100A8
(Fig. 9Ba and b). Underneath the scab containing
intensely stained S100A8-positive neutrophils, the
uninjured and proliferating keratinocytes were S100A8positive; superficial cornified keratinocytes were more
intensely positive, compared to controls (Fig. 9Ba
and c). In 7-day wounds, healing was evident, with
little inflammatory component, decreased ⁄ no vascularity and many fibroblasts aligned along collagen
fibers (Fig. 9Ca). Fibroblast-like cells were S100A8negative and keratinocytes were weakly positive
(Fig. 9Cb).

Discussion
Fibroblasts are ubiquitous sentinel cells that participate in local inflammatory processes by directly interacting with infiltrating leukocytes. Leukocytes residing
in, or recruited into tissues are surrounded by a meshwork of fibroblasts and both cell types produce, and
are influenced by mediators that regulate inflammation, wound healing, fibrosis, oxidative processes and
vascular remodeling. This study provides the first evidence that S100A8 is regulated in fibroblasts by particular growth factors, further supporting its role in
inflammatory processes.
FGFs stimulate proliferation of many cell types
involved in wound healing including endothelial cells,
fibroblasts and keratinocytes and are essential for survival, replication, differentiation and migration of various cell types during embryonic and fetal development
[42,43]. Interestingly, S100A8 has roles in cell migration [24,25] and S100A8, but not S100A9 [29,44], is
essential for embryonic development [45]. FGF-2
up-regulated S100A8, but not S100A9, mRNA in 3T3

and primary fibroblasts. A fragment of human S100A8
(S100A821)45) is chemotactic for fibroblast-like periodontal ligament cells [46] and this may be a function
of S100A8 released as a consequence of wound healing
and in an inflammatory environment. S100A8 induction was confluence-dependent (Fig. 1), a requirement
similar to its induction in microvascular endothelial cells [34]. Although fibroblasts do not normally
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F. Rahimi et al.

establish contacts in vivo, in culture they establish gap
junctions [37] indicating metabolic interdependence.
Cell–cell ⁄ cell–ECM contacts may provide additional
signals for optimal induction of the S100A8 gene
in vitro and because of the potentiation exhibited by
heparin, ECM heparan sulfate may be important.
FGFs bind heparin and heparan sulfates in positively
charged pockets that facilitate formation and stabilization of FGF-2 dimers and higher oligomers, resulting
in a more stable FGF–FGFR signaling complex [47].
Heparin also protects FGF against glycosylation [48]
and proteolysis and FGF binding to ECM increases its
immediate availability and durability of action [47].
Responses to FGF-2 were strongly up-regulated and
prolonged by heparin (Figs 1–3 and 6), but heparin
alone had no effect (Fig. 6A), implying facilitation at
the level of the FGF–FGFR interaction.
IL-1b has high amino-acid homology with FGF-2,
and is a potent inducer of the S100A8 gene in microvascular endothelial cells [34] and weak inducer in
macrophages [26]. IL-1b up-regulated S100A8 mRNA
in fibroblasts (Fig. 3) and exceptionally strong synergy
was observed when in combination with FGF-2 ⁄ heparin, obvious at the mRNA (Table 1) and protein level

(Figs 7 and 8). In contrast, LPS, IFNc and TNF,
which induce S100A8 in macrophages [26] and endothelial cells [34] only induced S100A8 mRNA when
used in combination, suggesting cooperative pathways.
IL-1b synergistically increased S100A8 mRNA in primary fibroblasts costimulated with FGF-2 ⁄ heparin
(Figs 3D and 6D). Because of functional and phenotypic heterogeneity [36,37], fibroblasts from various
sources were tested and because responses in primary
cells were relatively weak, quantitative RT-PCR was
used to detect the S100A8 gene and to assess S100A9
gene coinduction. The stability of S100A8 protein in
neutrophils is suggested to be dependent on S100A9
coexpression [29], but, like the situation in activated
murine macrophages [26,27] and keratinocytes [22],
S100A9 was not coinduced with S100A8 in any fibroblast type tested, strongly supporting our proposal that
S100A8 ⁄ S100A9 coexpression is not mandatory for the
function or stability of murine S100A8. This may not
be the case with the human proteins that are generally
coexpressed [49] although S100A8 alone was detected
in the human LDL proteome by mass spectrometry
and peptide mass fingerprinting [28].
Negligible S100A8 was secreted in response to IL-1b
and FGF-2 ⁄ heparin (Fig. 7A). This is in stark contrast
with activated macrophages which secrete high levels
in response to various stimulants, particularly LPS
with IL-10 or prostaglandin E2 [26,27], but similar to
IL-1-activated microvascular endothelial cells [34] and
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F. Rahimi et al.

UVA-irradiated keratinocytes [22], where the protein is
located in the nuclei and cytoplasm of a proportion of
cultured cells. The high levels of S100A8 mRNA found
in FGF-2 ⁄ heparin-activated fibroblasts costimulated
with IL-1b correlated well with protein levels detected
by western blotting (Fig. 7), and S100A8-positive
fibroblasts were more numerous (Fig. 8). S100A8
induced by FGF-2 ⁄ heparin ⁄ IL-1b was mainly cytoplasmic (Fig. 8F) with no obvious differences in distribution between cells with apparently pyknotic or
normal nuclei.
FGF-2 signals through the MAPK and extracellular
signal-regulated kinase 1 (ERK1, p44) and ERK2 (p42)
pathways [43]. Similarly, IL-1b activates MAPK and
stress-activated protein kinases which are involved in
the G0–G1 cell-cycle transition in human neuroma fibroblasts [50]. IL-1b can also activate p38 MAPK, protein
kinases A and C (PKC), phospholipase C, and signals
via nuclear factor jB [51]. The MAPK pathway is
important in cell proliferation, differentiation, embryogenesis and synthesis of inflammatory cytokines [43,52]
and is essential for S100A8 induction in macrophages
[27]. Inhibitors of the MEK and p38 pathways both
partially abrogated FGF-2 ⁄ heparin- and IL-1b-induced
expression in 3T3 fibroblasts (Fig. 5), indicating their
partial involvement. Induction of S100A8 mRNA by
FGF-2 was dependent on de novo protein synthesis
(Fig. 5). FGF-2 signaling can also occur via activation
of phospholipase C-c, PKC and increased Ca2+ flux
[43]. The S100A8 gene is also regulated by PKC- and
Ca2+-dependent mechanisms in macrophages [26,53],
and these results suggest complex control involving
converging pathways for its induction in fibroblasts.
Promoter deletion analyses indicated differential

regulation of S100A8 induction by IL-1b and FGF2 ⁄ heparin (Fig. 4). The minimal promoter required for
FGF-2- ⁄ heparin-induced responses was restricted to
the region from )94 to +465 (Fig. 4). Deletion of the
region from )178 to )94 bp, which contains activated
protein-1, Ets and C ⁄ EBP motifs, totally negated
activity induced by LPS ⁄ IL-10 in macrophages [27],
indicating distinct responsive elements activated by
FGF-2 ⁄ heparin in fibroblasts and LPS ⁄ IL-10 in macrophages. IL-1b-responsive elements may be distinct
from FGF-2-responsive elements as they were not
located within )917 to +465 bp and preliminary
experiments with a construct spanning 4.9 kb upstream
of the transcriptional start site strongly indicate differences in transcriptional regulation. The strong synergy
generated by the combination of FGF-2 ⁄ heparin and
IL-1b may be mediated through distinct enhancer ⁄
responsive elements in the two regions or IL-1b may
enhance S100A8 mRNA stability.
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Expression and regulation of S100A8 in fibroblasts

TGF-b accelerates deposition, remodeling and maturation of collagen later in the healing process of dermal wounds [54,55]. Differentiation of fibroblasts from
a proliferative phenotype to collagen-producing and
then to myofibroblastic phenotypes is believed to be
mediated by TGF-b [39]. IL-1b and FGF-2 promote
proliferation [56,57], whereas TGF-b promotes differentiation to myofibroblasts [39] and antagonizes the
IL-1- and FGF-2-induced proliferative phenotypes
[58,59]. Here we show that S100A8 mRNA (Fig. 6)
and protein (Fig. 7) induced by FGF-2 ⁄ heparin was
suppressed in fibroblasts costimulated with TGF-b,
possibly by decreasing S100A8 mRNA stability. Interestingly, induction by IL-1b was unaltered (Fig. 6).

The effect is similar to TGF-b inhibition of the FGF2-induced aA-crystallin promoter activity observed in
lenticular epithelial explants and TGF-b has been associated with pathological changes, including apoptosis
and accumulation of ECM, in some forms of cataract
[60]. The pattern of S100A8 gene regulation indicates
that this protein may be involved in fibroblast-to-myofibroblast differentiation at sites of inflammation and
repair ⁄ remodeling, particularly as S100A8 has been
associated with myeloid cell differentiation [26,61].
Moreover, S100A8 and S100A9 are expressed in
human and murine wounds and are associated with
keratinocyte proliferation ⁄ differentiation [21]. Whether
the TGF-b-induced myofibroblast phenotype correlates
with down-regulation of S100A8 is worthy of investigation, particularly as S100A8 expression in monocytes
disappears in tissue [61] and exudate macrophages [26].
These investigations are underway in our laboratory.
During the later stages of wound healing, TGF-b is
active and its receptors highly expressed whereas
FGF-2 activity decreases [54,55]. At this stage, fibroblast-derived S100A8 would be expected to be downregulated. This would be consistent with reduced
inflammation in the growing scar tissue and less oxidative stress as levels of tissue antioxidants including
glutathione, catalase, superoxide dismutase, glutathioneS-transferase, and glutathione peroxidase partially or
completely recover as healing progresses [62]. This
correlated well with the immunohistochemical studies
in the healing rat wound. Some spindle-shaped fibroblast-like cells in the granulation tissue were S100A8positive 2 and 4 days after dermal injury whereas
almost all fibroblasts were negative 7 days post injury
(Fig. 9).
The increasing importance of various S100 proteins
in regulating oxidative processes is emerging [33]
and in the mouse, S100A8, but not S100A9, was
induced in dermal keratinocytes following oxidative stress and by ultraviolet A irradiation, and gene
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Expression and regulation of S100A8 in fibroblasts

induction was dependent on generation of ROS [22].
Interestingly, under nonreducing ⁄ nondenaturing conditions, only mS100A8 dimer was detected in activated
fibroblast lysates (Fig. 7B), a structural modification
induced in S100A8 by oxidants such as peroxide [31],
suggesting a function partially analogous to S100A2
[63]. S100A2 also forms disulfide bonds in response to
H2O2 and is sensitive to early cellular responses to oxidative stress [33,63]. Redox-dependent signaling and
intracellular levels of ROS regulate cell-densitydependent growth [64,65] and increasing fibroblast
confluence concurs with augmented intracellular oxidative stress [65]. Interestingly, in lung fibroblasts, FGF-2
rapidly increases intracellular O2– without extracellular
H2O2 release whereas TGF-b promotes extracellular
H2O2 release that is O2– independent and NADHoxidase- ⁄ Ras-independent [66]. Because S100A8 is an
efficient scavenger of ROS [30,31] it may regulate
intracellular redox-mediated pathways involved in
fibroblast growth ⁄ differentiation. These pathways are
currently under investigation in our laboratory.

Experimental procedures
Reagents
Recombinant mS100A8 was generated from the glutathione-S-transferase fusion protein expressed in Escherichia
coli as described previously [67]. For production of polyclonal antibodies, S100A8 and mOxS100A8 (50 lg) [30]
bound to nitrocellulose particles in Freund’s complete adjuvant (Sigma, St Louis, MO, USA) were prepared as described [67], and injected intradermally into New Zealand
white rabbits. Rabbits were boosted after 4 and 8 weeks
with 100 lg mOxS100A8 or mS100A8 in incomplete Freund’s adjuvant (Sigma). IgG from pooled sera was purified
by Protein A-Sepharose (Amersham Pharmacia Biotech,
Buckinghamshire, UK). Titer and reactivities of IgG with
mS100A8 ⁄ mOxS100A8 were tested by immunoblotting and

ELISA as described [40]. Pre-immune sera did not react
with mS100A8 or mOxS100A8. Anti-mOxS100A8 and antimS100A8 did not cross-react with S100A9 monomer but
recognized the murine and rat S100A8 monomer and oxidized forms from 20 kDa to <100 kDa.
Cytokines, growth factors and chemicals were obtained
from the following sources: recombinant human FGF-2
and recombinant human TGF-b1 were from Sigma, murine
IL-1b from Genzyme (Cambridge, MA), and murine IFNc
from Genzyme, or Genentech, or Sigma. TNF was from
Genzyme or Sigma, LPS (E. coli, 055:B5) was from Difco
(Detroit, MI). The MAPK inhibitors, PD 098059 and SB
202190, Dulbecco’s phosphate-buffered saline (DPBS), collagenase,
N-2-hydroxyethylpiperazine-N¢-2-ethanesulfonic
acid (Hepes), sodium pyruvate, l-glutamine, penicillin,

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F. Rahimi et al.

streptomycin, trypsin-EDTA solution, Triton X-100, Tween
20, DTT, ovalbumin, BSA, saponin, normal goat serum,
agarose-bound caprine anti-rabbit IgG, ActD, cycloheximide, hematoxylin, c-methacryloxypropyltrimethoxysilane
(silane) and diaminobenzidine were from Sigma. RPMI
1640, Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS), and LipofectAMINE 2000 reagent
were from Gibco Invitrogen Corporation (Grand Island,
NY, USA), and bovine calf serum (BCS) from HyClone
Laboratories (Logan, UT, USA). Horseradish-peroxidase
(HRP)-conjugated caprine anti-rabbit IgG was from BioRad (Hercules, CA, USA), caprine anti-rabbit IgG-AlexaFluor-568 and the nucleus-specific counterstain, DAPI,
were form Molecular Probes (Eugene, OR, USA), Complete Protease Inhibitor cocktail tablets from Boehringer
Mannheim (Mannheim, Germany) and chemiluminescence

reagents from NEN Life Science Products (Boston, MA,
USA). b-mercaptoethanol was from ICN Biomedicals
(Aurora, OH, USA) and the Dual-Luciferase Assay System
from Promega. Proteinase K was obtained from MERCK
(Darmstadt, Germany).
DMEM, RPMI 1640, DPBS, CaCl2 solution, antibiotics
and b-mercaptoethanol [prepared in pyrogen-free distilled
water (Baxter Healthcare Pty. Ltd, N.S.W, Australia)] were
sterilized by filtration through Zetapor membranes (0.2 lm,
Cuno, Meriden, CT, USA) to remove contaminating
LPS. Other reagents and culture ware were sterile and
tissue-culture grade.

Isolation of primary cells, and cell culture
SPF were isolated and cultured as described [68] with some
modifications. Briefly, spleens of Quakenbush-Swiss mice
were removed, minced into small pieces, and digested for
20 min at 37 °C in collagenase (2 mgỈmL)1) in DPBS supplemented with CaCl2 (1 mm) then passed through a tissue
strainer (70 lm, BD Biosciences, San Jose, CA, USA), and
washed three times in RPMI 1640 containing 10% (v ⁄ v)
heated (56 °C, 30 min) FBS, Hepes (10 mm), sodium pyruvate (1 mm), l-glutamine (2 mm), 100 mL)1 penicillin,
100 lgỈmL)1 streptomycin, 2-mercaptoethanol (50 lm), and
NaHCO3 (19 mm), hereafter referred to as RPMI culture
medium (RPMI CM). Cells were resuspended in RPMI
CM in tissue-culture dishes (100 mm diameter) and maintained at 37 °C in humidified 5% CO2 in air. Non-adherent
cells and debris were removed by replenishing RPMI CM
after 24, 48 and 72 h. Cells were replenished with fresh
medium every 3 days until they reached confluence. SPF
from passages 2 through 8 were used in the experiments.
Primary fibroblasts obtained by this method do not contain

macrophages or other cells of hematopoietic origin [68] and
had typical fibroblast morphology.
In other experiments, BM cells were collected from
femurs of BALB ⁄ c mice and maintained in tissue-culture
flasks in RPMI CM. CM was replenished after 24, 48 and

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F. Rahimi et al.

72 h and nonadherent cells and debris were thereby
removed. The adherent BMF were maintained as for SPF
and used in experiments from passages two to eight.
NIH 3T3 cells (American Type Culture Collection) were
maintained for up to 15–20 passages in high-glucose
(4.5 gỈL)1) DMEM containing sodium pyruvate, supplemented with 44 mm NaHCO3, 2 mm l-glutamine, antibiotics and 10% heat-treated BCS, hereafter referred to as
DMEM CM.
When confluent, SPF, BMF, or 3T3 cells were rinsed
twice in Ca2+/Mg2+-free DPBS, and detached after incubation (2 min for 3T3, 5–7 min for primary cells, 37 °C) with
trypsin-EDTA solution (0.05 and 0.02% w ⁄ v, respectively)
diluted in Ca2+-free DPBS. RPMI CM or DMEM CM
was added to arrest digestion, cells harvested and seeded
onto tissue-culture flasks or 60-mm dishes (BD Biosciences)
for maintenance or stimulation (2–3 · 106 cells per dish).
Generally, cells grown to 100% confluence were activated
with the desired stimulants after a DPBS wash and replenishment with fresh CM. Cell viability by Trypan-blue exclusion was > 90–97%.
To determine whether de novo protein synthesis was
required for induction of S100A8 mRNA, confluent 3T3
cells were preincubated with the protein synthesis inhibitor,

cycloheximide (5–10 lgỈmL)1), for 4 h before stimulation
with activators and RNA analysis performed after 24 h.
For inhibition of RNA synthesis, fibroblasts were stimulated with particular stimulants for 20 h before an additional
20-h incubation with ActD (10 lgỈmL)1) and RNA levels
analyzed before and after ActD addition. To determine the
role of the MAPK pathway in S100A8 mRNA induction,
3T3 cells were preincubated for 4 h with inhibitors (PD
098059, the MEK inhibitor, 50–75 lm; SB 202190,
JNK ⁄ p38 inhibitor, 10–20 lm) before stimulation.

RNA extraction and Northern analysis
Total cellular RNA from 2.5 to 3.5 · 106 fibroblasts was
purified as described [69]. Murine BM RNA [67] was
used as a positive control. RNA (25–30 lg) was sizefractionated on 1.5% agarose gels containing 0.4 lgỈmL)1
ethidium bromide and transferred onto Hybond N+
membranes (Amersham) [69]. Pre-hybridizations were performed at 53 °C (2 h) in a formamide-containing buffer
[67]. 32P-labelled mS100A8 riboprobe and 18S rRNA
oligoprobe were prepared [34] and purified using ProbeQuant G-50 Micro Columns (Amersham). Blots were
hybridized at 53 °C overnight, washed [34] and exposed
to phosphoimager plates (Bio-Rad or FUJI Photo Film
Co, Ltd, Japan). Blots were scanned using the Molecular
Imager System (GS-525, Bio-Rad) and analyzed with
Multianalyst 1.0 software (Bio-Rad). Alternatively, the
FLA5000 imaging system (FUJI) and the L Process and
Image Gauge software packages were used. The relative magnitude of expression of S100A8 mRNA was

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Expression and regulation of S100A8 in fibroblasts

determined using quantitative densitometry, normalized to
the level of 18S rRNA on the same blot and expressed
as the percent maximum mRNA level.
In some experiments, total RNA was subjected to quantitative RT-PCR using the primers described [6]. cDNA levels during the linear amplification phase were normalized
against hypoxanthine phosphoribosyl-transferase (HPRT)
controls. Assays were in triplicate and mean ± standard
deviation (SD) determined.

Reporter assays
The mS100A8-promoter-luciferase-fused reporter plasmids
were described previously [27]. Sub-confluent 3T3 cells
grown in 24-well plates were transiently transfected with
0.8 lg firefly luciferase constructs, or the parent plasmid
pGL2-basic DNA, or the pGL2 promoter DNA combined with 0.03 lg of the control pRL-TK plasmid
(Promega) in the presence of LipofectAMINE. After
24 h, cells were replenished with fresh DMEM CM and
stimulated with FGF-2 (1.5 nm) + heparin (1 ImL)1)
or with IL-1b (10 mL)1) for another 48 h. Firefly and
Renilla luciferase activities in cell extracts (20 lL) were
assayed using the Dual-Luciferase Assay System in a TD20 ⁄ 20 Luminometer (Turner Design, Sunnyvale, CA,
USA) following manufacturer’s instructions. Promoter
activity was normalized to Renilla luciferase which resulted in reproducible and constant values relative to pGL2
promoter.

Protein purification and Western blotting
Control or stimulated (28–30 h) 3T3 cells (20–25 · 106)
were washed twice with DPBS after removal of supernatants, lysed in DPBS containing 1% (v ⁄ v) Triton X-100,
50 mm Tris ⁄ HCl, pH 8.0 and Complete Protease Inhibitors,
subjected to three freeze-thaw cycles and sonicated on ice
for 2 min. Debris was removed by centrifugation and

lysates (5 mL) and supernatants (9 mL) concentrated to
1 ⁄ 5th starting volume in a vacuum lyophilizer. Following
dialysis overnight at 4 °C in water (1 L) to remove excess
salt, samples were incubated with anti-mOxS100A8
(10 lgỈmL)1) overnight at 4 °C, then with agarose-bound
caprine anti-rabbit IgG slurry (70 lLỈmL)1) overnight at
4 °C. Beads were sedimented (1600 g), washed thrice with
lysis buffer, boiled (100 °C, 5 min) in Tricine sample buffer
(50 lL, Bio-Rad), and 20 lL separated by SDS ⁄ PAGE as
described previously [67], with DTT (100 mm). Alternatively, supernatants and lysates were directly adsorbed with
Sepharose-coupled anti-mS100A8, washed with phosphatebuffered saline (NaCl ⁄ Pi) and bound proteins eluted with
0.2 m glycine ⁄ 1 m NaCl (pH 3.0; 0.5 mL), and pH adjusted
with 100 lL Tris ⁄ HCl (pH 9.0). Eluates (600 lL) were
directly subjected to C4 reverse-phase HPLC [31] prior to
nonreducing SDS ⁄ PAGE and Western blotting [67].

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Expression and regulation of S100A8 in fibroblasts

Protein localization
Confluent 3T3 cells in Lab-Tek Permanox chamber slides
(Nalge Nunc International, Roskilde, Denmark) were activated with stimulants for 30 h, rinsed thrice in NaCl ⁄ Pi,
slides air-dried then re-hydrated in NaCl ⁄ Pi (10 min), fixed
in 4% paraformaldehyde (10 min) and permeabilized
(10 min) with 0.5% saponin ⁄ 0.1% BSA in NaCl ⁄ Pi. After
blocking with normal goat serum (20% w ⁄ v in saponin–
BSA solution, 20 min), and incubation with antimOxS100A8 IgG or preimmune rabbit IgG (both at
10 lgỈmL)1 in saponin–BSA) overnight at 4 °C, slides were

rinsed in NaCl ⁄ Pi and incubated with anti-rabbit IgGAlexa-Fluor-568 (1 : 200, in saponin–BSA) for 1 h in the
dark at 25 °C, rinsed in NaCl ⁄ Pi (3 · 5 min) and nuclei
stained with DAPI (0.3 nm in NaCl ⁄ Pi, 10 min in the dark).
Slides were mounted in Vectashield Mounting Medium
(Vector Laboratories, Burlingame, CA, USA), cover-slipped
and examined using a Leica DM IRB inverted confocal
microscope attached to a Leica TCS SP scanner (Leica
Microsystems, Mannheim, Germany). Images were captured using Leica Confocal Software and processed using
Adobe Photoshop 7.0.
For immunohistochemistry, paraffin-embedded tissue
blocks of dermal wounds from three rats wounded by scalpel incision under anesthesia, sutured and dressed, were
provided by Professor Rolfe Howlett. Rats were sacrificed
2, 4 and 7 days after wounding and fixed tissue processed,
sectioned (5 lm) and mounted onto silane-coated slides; all
experiments were conducted according to local ethics committee approval. Deparaffinized sections were re-hydrated
then washed in NaCl ⁄ Pi ⁄ Triton X-100 (0.3% v ⁄ v, 15 min),
treated with 0.2 m HCl (15 min), permeabilized with proteinase K (25 lgỈmL)1) in 0.1 m Tris ⁄ HCl ⁄ 50 mm EDTA,
pH 8.0 (37 °C, 30 min), washed with 0.2% (w ⁄ v) glycine
followed by NaCl ⁄ Pi, and fixed with 4% paraformaldehyde
in NaCl ⁄ Pi (5 min). Sections were blocked sequentially with
H2O2 (3% v ⁄ v in NaCl ⁄ Pi, 30 min) and 10% normal
goat serum (30 min), incubated for 1 h (25 °C) with rabbit
anti-mS100A8 IgG or nonimmune rabbit IgG (both
10 lgỈmL)1) in NaCl ⁄ Pi containing 0.05% saponin ⁄ 0.1%
BSA. Optimal IgG concentrations were predetermined by
titration. Slides were washed with NaCl ⁄ Pi and bound antibody detected after 30-min incubation with biotinylated
caprine anti-rabbit IgG (Dako) and streptavidin-peroxidase
(Kirkegaard & Perry Laboratories, Inc. Gaithersburg, MA,
USA). Visualization was with diaminobenzidine preceding
counterstaining with hematoxylin. For other controls, primary or secondary antibodies were omitted.

Acknowledgements
The authors are obliged to Professor Rolf Howlett for
the preparation and donation of wound specimens
and Drs M. Raftery, R. Passey, N. Tedla, Z. Yang,

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F. Rahimi et al.

W. X. Yan and H. Cai for technical help ⁄ advice.
Funding from the National Health and Medical
Research Council of Australia is acknowledged; FR
held an Australian Postgraduate Award.

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