Full text of DOI:10.1038/sj.bjp.0704769

British Journal of Pharmacology (2002) 136, 793 ± 801
ã
2002 Nature Publishing Group All rights reserved 0007 ± 1188/02 $25.00
www.nature.com/bjp
Oncostatin M stimulates proliferation, induces collagen production
and inhibits apoptosis of human lung ®broblasts
1
,2
1,3
1,2
Amelia K. Scadi, Steven E. Mutsaers, Yuben P. Moodley, Robin J. McAnulty,
3
3
1,2
Geo€rey J. Laurent, Philip J. Thompson & * Darryl A. Knight
1,2
1
Asthma and Allergy Research Institute, Ground Floor, ``E'' Block, Sir Charles Gairdner Hospital, Verdun Street, Nedlands,
Western Australia 6009; Department of Medicine, The University of Western Australia, Nedlands, Western Australia, 6009
2
3
and Centre for Cardiopulmonary Biochemistry, University College London Medical School, Rayne Institute, London WCIE
6
JJ
1
Oncostatin M (OSM), a member of the interleukin-6 (IL-6) cytokine family, acts on a variety of
cells and elicits diversi®ed biological responses, suggesting potential roles in the regulation of cell
survival, di€erentiation and proliferation.
2
collagen production and spontaneous apoptosis. The proliferative e€ects of OSM (0.5 ± 100 ng ml
We have examined the e€ect of OSM on the regulation of human lung ®broblast proliferation,
1
7
)
were assessed using a MTS assay as well as [ H]-thymidine incorporation and cell counts at 24 and
3
4
8 h. Hydroxyproline was measured as an index of procollagen production by high pressure liquid
chromotography (HPLC). Apoptosis was determined by annexin staining.
OSM enhanced the mitotic activity of lung ®broblasts in a time and dose dependent manner.
Maximum proliferation of 57% above control was observed after incubation for 48 h with
ng ml OSM (P50.05).
Incubation with the mitogen activated protein kinase (MAPK) kinase inhibitor, PD98059 or the
3
71
2
4
tyrosine kinase inhibitor, genestein both signi®cantly reduced the mitogenic e€ect of OSM (P50.05).
In contrast, proliferation in response to OSM was not regulated by induction of cyclo-oxygenase
and subsequent prostaglandin E (PGE ) release or by IL-6.
5
2
2
6
OSM also stimulated ®broblasts to synthesize pro-collagen by a maximum of 35% above control
levels after 48 h (P50.05).
7
8
OSM signi®cantly inhibited the spontaneous apoptosis of ®broblasts at 24 and 48 h.
These results provide evidence that OSM has pro-®brotic properties and suggest that it may play
a role in normal lung wound repair and ®brosis.
British Journal of Pharmacology (2002) 136, 793 ± 801
Keywords: Cytokine; asthma; lung; tissue repair; ®brosis
Abbreviations: BSA, bovine serum albumin; COX, cyclo-oxygenase; DAPI, 4,6 diamidino-2-phenylindole; EIA, enzyme
immunoassay; ELISA, enzyme-linked immunosorbent assay; ECM, extracellular matrix; FCS, foetal calf serum;
3
[
H]-TdR, tritiated thymidine; HPLC, high pressure liquid chromatography; hyp, hydroxyproline; IL-6,
interleukin-6; LIF, leukaemia inhibitory factor; MAPK, mitogen activated protein kinase; MEK, mitogen
activated protein kinase kinase; MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazolium
inner salt); OSM, Oncostatin M; OSMR, OSM receptor; PBS, phosphate-bu€ered saline; PI, propidium iodide;
PGE , prostaglandin E ; TGFb, transforming growth factor beta; TIMP-1, tissue inhibitor of metalloprotease
2
2
71; TTBS, tris-bu€ered saline/Tween-20
Introduction
Chronic in¯ammatory airway diseases such as asthma and
pulmonary ®brosis are characterized by an increased
deposition of extracellular matrix (ECM), concomitant with
proliferation and activation of sub-epithelial ®broblasts
in¯ammatory cytokines such as interleukin-1 beta (IL-1b),
tumour necrosis factor alpha and anti-in¯ammatory cyto-
kines such as macrophage inhibitory protein (Chung &
Barnes, 1999). Fibroblasts also release multiple growth
factors such as transforming growth factor beta (TGFb),
which directly in¯uence the deposition and composition of
the ECM (Chung & Barnes, 1999) and therefore may control
multiple processes in wound repair (Border & Noble, 1994).
However the regulatory role and relative importance of other
more recently identi®ed cytokines is far from clear and needs
to be assessed.
(
Je€ery, 1991; Brewster et al., 1990; Gizycki et al., 1997).
Fibroblasts are important sources of cytokines, growth
factors and mediators that can in¯uence the behaviour of
adjacent cell types and thereby contribute to the initiation
and resolution of airway in¯ammation. These include pro-
*
Author for correspondence at: Asthma and Allergy Research
OSM is a pleiotropic cytokine that was ®rst described as a
product of in¯ammatory cells such as T-lymphocytes,
monocyte/macrophages and more recently neutrophils (Rose
Institute, Ground Floor, ``E'' Block, Sir Charles Gairdner Hospital,
Verdun Street, Nedlands, Western Australia 6009
E-mail: dknight@cyllene.uwa.edu.au

794
A.K. Scaffidi et al
OSM exerts multiple effects on fibroblast function
&
Bruce, 1991; Zarling et al., 1986). OSM is a member of the
(Life Technologies, Melbourne, VIC, Australia) supplemen-
ted with 10% foetal calf serum (FCS), 2 mM L-glutamine,
IL-6 family of cytokines that also includes leukaemia
inhibitory factor (LIF), interleukin-11, ciliary neurotrophic
factor and cardiotrophin-1 (Taga, 1996; 1997). These
cytokines produce a variety of overlapping and speci®c
biological e€ects related to the regulation of in¯ammation.
Recent studies have examined the localization of these
cytokines and their role in airway in¯ammation (Knight et
al., 1999a; b). To date OSM has been shown to upregulate
the production of lung epithelial cell anti-proteases (Cichy et
71
100 mg ml penicillin and gentamycin. Cells were kept in a
humidi®ed atmosphere of 5% CO in air at 378C. Medium
was routinely changed every 3 to 4 days.
2
Analysis of OSMR expression
Flow cytometry was used to con®rm the presence of OSMR
6
71
on the cell surface. Fibroblasts (1610 ml ) were cultured in
six well culture plates in 0.4% FCS/DMEM and allowed to
adhere for 16 h as described above. Cells were detached from
the well with either Trypsin or EDTA and resuspended in
serum free DMEM supplemented with 1% BSA. Cells were
washed three times in PBS (phosphate-bu€ered saline)
containing 0.1% Tween-20 incubated with rabbit anti-human
OSMR (1 : 1000) before incubation with FITC-conjugated
goat a rabbit IgG (1 : 500) at 48C for 30 min. Cells were
washed, resuspended in serum free DMEM and analysed
using a FACScan ¯ow cytometer.
2
al., 1998) and PGE (Knight et al., 2000) as well as the
production of TIMP-1 in human lung ®broblasts (Richards et
al., 1993). In dermal ®broblasts, OSM is an activator of the
collagen a2 (1) promoter (Ihn et al., 1997) and potently
induces collagen and glycosaminoglycan production (Duncan
et al., 1995). Furthermore, mice with targeted overexpression
of OSM to the pancreas, developed severe localized ®brotic
lesions (Bamber et al., 1998). These studies suggest that this
cytokine may have wound healing or pro®brotic properties.
In the current study we have examined the e€ects of OSM
on human lung ®broblast proliferation, pro-collagen synthesis
and spontaneous apoptosis. The results demonstrate that
OSM is a mitogen for these cells, induces procollagen
production and is anti-apoptotic. Taken together, these
results suggest that OSM may play an important role in the
wound healing response, asthma and ®brosis.
Cell proliferation assays
Fibroblasts were seeded in DMEM/10% FCS at a density of
4
3610 cells per well in 24 well tissue culture plates and
allowed to adhere for 24 h. The cells were the quiesced by
replacing the medium with serum free DMEM for a further
4 h before being treated with either OSM at 0.5 ±
2
71
Methods
100 ng ml at 378C for 24 or 48 h. Experiments were also
performed in the presence of pharmacological inhibitors.
These inhibitors were added 30 min prior to the addition of
Cytokines and chemicals
71
OSM (2 ng ml ). At the end of each incubation period,
proliferation was assessed using an MTS assay. This assay
measures mitochondrial activity through the formation of an
insoluble formazan salt and has been shown to correlate to
cell density (Martinez et al., 1999). MTS was diluted 1 : 20
with serum free DMEM and 300 ml was added to each well.
After 1 h incubation, cell proliferation was assessed at
Recombinant human OSM and anti-gp130 antibody were
purchased from R&D systems (Minneapolis, MN, U.S.A.).
TGFb1, indomethacin, DAPI, Propidium Iodide (PI) and rat-
tail collagen were purchased from Sigma (Castle Hill, NSW,
Australia). IL-1b and Annexin V-FITC were purchased from
Boehringer Mannheim (Melbourne, Vic, Australia). Genestein
and PD98059 were purchased from Calbiochem (La Jolla, CA,
U.S.A.). Nimesulide and monoclonal antibodies recognizing
COX-1 and COX-2 were purchased from Cayman Chemical
Co (Ann Arbor, MI, U.S.A.). Peroxidase-conjugated second-
ary antibodies were purchased from Dako (Carpintaria, NSW,
Australia). Alexa-¯uor conjugated secondary antibodies were
purchased from Molecular Probes (Brisbane, QLD, Austra-
lia). Monoclonal antibodies recognizing cyclin DI and IL-6
were purchased from Santa Cruz and Pharmingen (La Jolla,
CA, U.S.A.). MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2H-tetrazolium, inner salt)] was purchased
450 nm in
Devices Co., CA, U.S.A.). Results were expressed as
absorbance or as percentage change in absorbance
a Spectramax spectrophotometer (Molecular
a
compared to control (i.e. cells with medium alone). Direct
3
cell counts and tritiated thymidine ([ H]-TdR) incorporation
were also performed to con®rm the results of the MTS assay.
3
[ H]-TdR incorporation assays were performed by adding
3
71
[ H]-TdR (1 mCi ml ) to each well 6 h after the addition of
treatments. The cells were incubated at 378C for 18 h. Cells
were then harvested using a cell harvester and radioactivity
incorporation assessed using a Packard direct b counter
(Canberra, NSW, Australia). For cell counts, cells were
stained with Trypan blue and the number of viable cells
counted using a Neubauer Haemocytometer.
3
from Promega (Madison, WI, U.S.A.). [ H]-thymidine was
purchased from Amersham (Sydney, NSW, Australia). The
polyclonal antibody-OSMR antibody was a kind gift from
Immunex Corp. (Seattle, WA, U.S.A.).
IL-6 production
Cell culture
Following exposure of ®broblasts to OSM, supernatants were
analysed for IL-6 using a speci®c enzyme-linked immunosor-
bent assay (ELISA). Brie¯y, 96 well plates (Maxisorp, Nunc)
The normal diploid human foetal lung ®broblast cell line was
obtained from American Type Culture Collection (ATCC,
Rockville, MD, U.S.A) and was used between passages 11
and 24. Primary human lung ®broblasts were established
from explant cultures (Keerthisingam et al., 2001). Cells were
cultured in T-75 tissue culture ¯asks with DMEM medium
71
were coated with mouse anti-human-IL-6 (500 ng ml
0.1 M NaHCO /NaCO
night at 48C. After rinsing in PBS containing 0.5% v v
Tween-20, wells were blocked by addition of 1% (w v
in
bu€er, pH 9.6) and incubated over-
3
3
71
71
)
British Journal of Pharmacology vol 136 (5)

A.K. Scaffidi et al
OSM exerts multiple effects on fibroblast function
795
BSA in PBS/Tween-20. After multiple washings, 100 ml of
sample were added and the plate allowed to incubate at 48C
overnight. After washing, a biotinylated anti-mouse IgG
antibody was added and the plate allowed to incubate at
room temp for 1 h. Peroxidase-labelled streptavidin (S-HRP)
was then added for a further 30 min and the plates washed
three times in PBS/Tween-20. Peroxidase substrate (K-blue
ELISA substrate) was added to each well and the reactions
terminated by addition of 1 M phosphoric acid. Absorbances
were read at 450 nm on an ELISA plate reader Spectramax
immediately analysed on a FACScan ¯ow cytometer using
CellQuest software (Becton Dickinson, San Jose, CA,
U.S.A.). Ten thousand cells were acquired. Apoptotic cells
stained positively for Annexin V (AV) but excluded PI
+
7
(AV PI ), whilst necrotic cells were double positive
+ +
(AV PI ).
Determination of fibroblast procollagen production
Fibroblast procollagen production was assessed in vitro by
quantitating hydroxyproline (hyp) using reverse-phase high
pressure liquid chromatography (HPLC) as previously
described (Campa et al., 1990; Chambers et al., 1998).
Brie¯y, cells were grown to con¯uence in 2.4 cm diameter
wells in DMEM supplemented with 10% FCS and pre-
250 (Molecular Devices Co., CA, U.S.A.) at 450 nm. The
detection limit of the assay was 15 pg ml
71
.
PGE enzyme immunoassay
2
71
PGE was measured using a competitive enzyme immunoas-
2
incubated in DMEM supplemented with 1% FCS, 50 g ml
ascorbic acid (BDH, Poole, U.K.) and 0.2 mM proline
say according to the manufacturer's instructions (Cayman
Chemical, Ann Arbor, MI, U.S.A.). Plates were read at
(Sigma) at 378C for 24 h. The cells were then exposed to
71
71
4
(
50 nm using an ELISA plate reader Spectramax 250
Molecular Devices Co., CA, U.S.A.). The manufacturer's
speci®cations for this assay include an intra-assay coecient
of variation of P510%, cross reactivity with PGD and
PGF2a of less than 1% and linearity over the range of 10 ±
10 and 100 ng ml OSM or 1 ng ml TGF-b1 in similar
1% FCS supplemented culture medium (n=10 for each dose:
four for cell counts and six for hyp measurement) for a
further 48 h. For hyp measurement, the cell layer was scraped
into the culture medium and the contents of each well
collected. Wells were washed with 1 ml PBS and combined
with the original medium. Proteins were precipitated by the
2
1
1
000 pg ml
71
Western blotting
addition of ethanol to a ®nal concentration of 67% (v v ) at
4
8C overnight. Precipitated proteins were separated from free
The e€ect of OSM on the expression of COX-1 or COX-2 in
®broblasts was assessed by Western analysis. Trypsinized cells
amino acids by ®ltration through a 0.45 mm pore-size ®lter
(Millipore, Watford, Herts, U.K.) and hydrolyzed in 6 N
HCl at 1108C for 16 h. Hydrolysates were mixed with
activated charcoal, ®ltered and hyp quanti®ed using reverse-
phase-HPLC (Beckman, System Gold, High Wycombe,
Bucks., U.K.) after derivatization with 7-chloro-4-nitroben-
zofuran (Sigma) as previously described (Chambers et al.,
1998). The hyp content in each sample was determined by
comparing peak areas of samples from the chromatogram to
those generated from standard solutions. All values obtained
were corrected for the amount of hyp measured in the cell
layer and culture medium at the onset of the incubation
period and for cell number. Procollagen production is
6
(
0
2610 ) were incubated with lysis-bu€er (50 mM Tris,
.5 mM EGTA, 150 mM NaCl, 1% Triton X-100, pH 7.5)
containing a cocktail of protease inhibitors (Sigma), passed
4
through a ®ne gauge needle and centrifuged at 10 6g for
20 min. The protein content of the resultant supernatant was
determined using the Bradford method (BioRad). Equal
amounts of protein (40 mg) were added to SDS-sample bu€er
(
0.5 M Tris-HCl, 1% glycerol, 0.5% bromophenol blue, 0.5%
b-mercaptoethanol), boiled for 5 min and electrophoresed
through 12% polyacrylamide gel. Proteins were electroblotted
onto PVDF membranes (BioRad, Sydney, NSW, Australia),
which were blocked overnight in TTBS (tris-bu€ered saline/
Tween-20) containing 5% skim milk. Membranes were
probed with a mouse monoclonal antibody to human
COX-1 or COX-2 for 1 h at a dilution of 1 : 1000. After
repeated washes in TTBS, horse radish-peroxidase (HRP)-
conjugated anti-mouse IgG antibody (1 : 1000) was added for
6
71
expressed as nM of hyp 10 cells
.
1
h. After further washing in TTBS, blots were developed
with the ECL detection system and exposed to ECL-
Hyper®lm.
Analysis of apoptosis
2
Following 24 or 48 h incubation (378C, 5% CO in air) with
71
and without OSM (2 ng ml ), ®broblasts were harvested and
stained for the di€erential analysis of apoptotic and necrotic
cells according to the manufacturer's protocol (Boehringer
Mannheim). Brie¯y, 20 ml each of Annexin V-FITC and PI
7
1
(
(
50 mg ml ) was added per 1 ml of labelling bu€er
7
1
71
71
10 mmol l
HEPES, 140 mmol l
NaCl, 5 mmol l
Figure 1 Detection of the OSMR on lung ®broblasts. Cells were
stained with no antibody or with anti-OSMR followed by FITC-
labelled goat anti-rabbit IgG. Immunostaining was then analysed by
¯ow cytometry.
CaCl
broblasts (2610 per tube) and incubated in the dark (room
temp, 50 min). Fibroblasts were washed in PBS and
2
, pH 7.4). Labelling solution (100 ml) was added to
5
®
British Journal of Pharmacology vol 136 (5)

796
A.K. Scaffidi et al
OSM exerts multiple effects on fibroblast function
Statistical analysis
multiple comparisons. A P value of 50.05 was considered
signi®cant.
Data were expressed as mean+s.e.mean. Statistical compar-
isons of mean data were performed using a statistical t-test or
a one-way ANOVA with post-hoc Bonferroni correction for
Results
Detection of OSMR on lung fibroblasts
FACS analysis with speci®c OSMR antibodies shows that
®broblasts express speci®c OSMR on their cell surface
(
Figure 1).
Effect of OSM on the proliferation of lung fibroblasts
OSM increased the mitotic activity of ®broblasts in a time
and dose dependent manner with a maximal response of 57%
71
above control at 2 ng ml after 48 h (Figure 2A) (P50.05).
These ®ndings were consistent with those obtained either by
3
[
H]-TdR incorporation (Figure 2B) and direct cell counting
Table 1). Proliferative responses to OSM were completely
(
abrogated by pre-incubating the cells with speci®c neutraliz-
ing antibodies against OSMR or gp 130 (Figure 2C).
Fibroblasts were also stained with antibodies to cyclin D1
7
1
(
Figure 3). Incubation of ®broblasts with OSM (2 ng ml
induced an increase in the number of cyclin D1 positive cells
Figure 3E) compared to unstimulated ®broblasts (Figure
D).
)
(
3
Figure 2 Proliferation of human lung ®broblasts in response to
OSM. Cells were exposed to OSM at concentrations ranging from
Figure 3 Detection of cyclin D1 expression in lung ®broblasts.
Immunohistochemical staining for cyclin D1 was performed on
unstimulated cells (D) or cells exposed to OSM (2 ng ml ) for 24 h
71
71
0
.5 ± 100 ng ml for 24, 48 or 72 h. Proliferative e€ect was examined
3
using an MTS assay (A) and con®rmed by [ H]-TdR uptake (B). The
proliferative e€ect of OSM was completely abrogated by incubation
of cells with neutralizing antibodies against OSMR and gp 130.
Results are means of four separate experiments performed in
triplicate. *P50.05 compared to cells in serum free conditions.
(E). The speci®city of the cyclin D1 antibody was con®rmed by
substituting the primary antibody with non-immune mouse IgG1 (F).
Cells were also stained with DAPI in order to enumerate cell nuclei
(A ± C). Immuno¯uorescent images from 1 mm sections were obtained
by scanning laser confocal microscopy at 406magni®cation.
British Journal of Pharmacology vol 136 (5)

A.K. Scaffidi et al
OSM exerts multiple effects on fibroblast function
797
Therefore the e€ect of the tyrosine kinase inhibitor
genestein (10 mM) on OSM-induced mitogenesis was inves-
tigated. Incubation of ®broblasts with genestein completely
abolished the proliferative e€ects of OSM (P50.05) (Figure
4
). In order to examine the role of p42/p44 MAPK in
OSM-induced proliferation, ®broblasts were treated with the
MEK inhibitor PD98059 (50 mM) for 1 h before the
addition of OSM for 24 and 48 h. This concentration is
consistent with the IC50 values of PD98059 for MEK1
(4 mM) and MEK2 (50 mM) in other cell systems (Xiao et
al., 2001). The proliferative responses of these cells were
reduced compared to OSM alone at both time points
Figure 4). However the proliferative e€ects of OSM was
(
not reduced to the same degree when the tyrosine kinase
pathway was abolished.
Effect of inhibition of COX-2 and PGE release on
2
OSM-induced proliferation
Figure 4 E€ects of the tyrosine kinase inhibitor (genestein) and
p42/44 MAPK inhibitor (PD98059) on OSM-induced proliferation.
Fibroblasts were seeded in 24 well plates and incubated with
genestein (10 mM) or PD98059 (50 mM) in the presence or in the
OSM has been shown to be a COX-2 dependent mitogen for
vascular smooth muscle cells (Bernard et al., 1999), while in
71
absence of 2 ng ml
OSM. At 24 and 48 h cell numbers were
assessed using an MTS assay. Results are means of four separate
experiments performed in triplicate. *P50.05 versus cells exposed to
other cells, PGE
(
2
is a negative regulator of cell growth
71
OSM 2 ng ml . #P50.05 compared to 24 h time point.
Belvisi et al., 1998). Thus, COX-2 expression and PGE2
release in response to OSM were investigated by EIA and
Western blotting. The non-selective COX inhibitor indo-
methacin (5 mM) and the COX-2 selective inhibitor, nimesu-
lide (5 mM) had no e€ect on ®broblast proliferation alone and
did not alter OSM-induced proliferation (Figure 5a).
Furthermore, OSM did not appear to induce the expression
Table
incubation with OSM
1 Cell counts of human lung ®broblasts after
OSM
(2 ng ml
OSM
(20 ng ml71)
71
Treatment
Control
)
of COX-1 or COX-2 (Figure 5b) or the production of PGE
Figure 5c).
2
(
2
4
4 h
8 h
2.51+0.29
3.50+0.65
4.25+0.48
5.25+0.25
3.0+0.41
4.0+0.58
Effect of endogenous IL-6 production on OSM-induced
proliferation
Cells were stained with Trypan blue and the number of
viable cells counted using a Neubauer haemocytometer and
6
is presented as 10 cells
s.e.mean of four independent experiments.
71
. Values represent means+
In other cell types OSM is a potent regulator of IL-6
production (Bernard et al., 1999; Brown et al., 1991) and in
turn IL-6 has been shown to be a mitogen for human
®
broblasts (Roth et al., 1995). Exposure of ®broblasts to
Table 2 Spontaneous apoptosis of human lung ®broblasts
after incubation with OSM
OSM for either 24 or 48 h did not evoke the release of
measurable quantities of IL-6 (data not shown).
Treatment
Control (%)
OSM (%)
Effect of OSM on spontaneous apoptosis
2
4
4 h
8 h
9.3+1.2
16.3+3.6
7.3+0.6*
9.9+1.79*
Human lung ®broblasts were incubated with OSM
71
Fibroblasts were harvested and stained for the di€erential
analysis of apoptotic and necrotic cells using Annexin V-
FITC and PI. Fibroblasts were washed in PBS and
immediately analysed on a FACScan ¯ow cytometer. Ten
thousand cells were acquired. Apoptotic cells stained
(20 ng ml ) for 24 or 48 h. Using Annexin V, we con®rmed
that at these time points OSM signi®cantly decreased the
percentage of spontaneous apoptotic cells (Table 1). At 24 h
spontaneous apoptosis was 9.3+1.2% in untreated cells and
7.4+0.6% in the OSM treated cells. At 48 h, spontaneous
apoptosis had increased to 16.3+3.6% in control cells,
compared to 9.9+1.79% in the OSM treated cells (P50.05).
The number of necrotic ®broblasts tended to increase with
time in culture, but represented less than 5% of the total cell
number in all experiments (data not shown).
+
7
positively for Annexin V (AV) but excluded PI (AV PI ),
+
+
whilst necrotic cells were double positive (AV PI ). Values
are presented as percentage of apoptotic cells and
represent mean+s.e.mean of four separate experiments.
P50.05 versus control.
a
*
The effect of OSM on procollagen production
Effects of p42/44 MAPK and tyrosine kinase inhibition on
OSM-induced proliferation
The e€ect of OSM on ®broblast procollagen production was
assessed by measuring hyp levels using HPLC. The
concentrations of OSM used were those previously shown
to be optimal for procollagen production by mesenchymal
OSM induces cellular responses via tyrosine phosphorylation
of multiple intracellular proteins (Heinrich et al., 1998).
British Journal of Pharmacology vol 136 (5)

798
A.K. Scaffidi et al
OSM exerts multiple effects on fibroblast function
Figure 5 E€ects of the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor nimesulide on OSM-induced
proliferation, COX expression and PGE release. (a) Cells were treated with indomethacin (5 mM) or the selective COX-2 inhibitor
nimesulide (5 mM) prior to incubation with OSM (2 ng ml ). Proliferative responses to OSM were not in¯uenced by either drug at
2
71
7
1
2
1
4 or 48 h. Detection of COX-1 and COX-2 protein. Fibroblasts were incubated for either 6 or 24 h with OSM (2 ng ml ) or IL-
71
b (2 ng ml ) and expression of both COX-1 and COX-2 was determined by Western analysis (b). PGE release in response to
2
7
1
OSM. PGE
2
release was measured by EIA in supernatants taken from ®broblasts treated with OSM (2 ng ml ) for 24 and 48 h (c).
Results are shown as means of four separate experiments. *P50.05 compared to cells in serum free medium.
cells (Duncan et al., 1995; Levy et al., 2000). OSM increased
1999) and vascular smooth muscle cells (Grove et al., 1993).
Furthermore, OSM stimulates extracellular matrix produc-
tion in dermal ®broblasts, hepatic stellate cells and in the
pancreas (Duncan et al., 1995, Ihn et al., 1997; Bamber et al.,
1998; Levy et al., 2000) suggesting that it may be involved in
a variety of ®broproliferative diseases. Whether OSM has
similar e€ects on human lung ®broblasts is unknown. In the
current study, we have demonstrated that OSM, acting
through its speci®c type II receptor (1) is a mitogen, (2) is
anti-apoptotic (3) stimulates collagen production from human
lung ®broblasts.
71
procollagen production at 10 and 100 ng ml by 35+2%
and 22+2% respectively above control although this increase
71
was only signi®cant at 10 ng ml (P50.05; Figure 6). For
71
comparison, an optimal stimulatory dose of 1 ng ml TGFb
1
was used as a positive control and stimulated ®broblast
procollagen production by 224+10% (P50.05).
Discussion
OSM is a growth regulatory cytokine and is synthesized by a
number of circulating in¯ammatory cells, including T-
lymphocytes, macrophages and neutrophils (Rose & Bruce,
OSM has profound e€ects on the behaviour of dermal
®broblasts (Ihn & Tamaki, 2000) as well as synovial and lung
®broblasts (Richards et al., 1997), suggesting that OSMR are
present on ®broblasts. However, OSM has also been shown
to induce cellular responses by engaging and activating the
LIFR, which has also been demonstrated on human lung
®broblasts (Knight et al., 1999b; Mosley et al., 1996). It was
therefore important to con®rm the presence of OSMR on
1
991; Grenier et al., 1999). Although OSM has been shown
to inhibit the proliferation of cells derived from solid tumours
as well as normal and malignant mammary epithelium (Liu et
al., 1998), it is also a potent mitogen for dermal ®broblasts
(Ihn & Tamaki, 2000), endothelial cells (Pourteau et al.,
British Journal of Pharmacology vol 136 (5)

A.K. Scaffidi et al
OSM exerts multiple effects on fibroblast function
799
Bornfeldt et al. (1997) recently demonstrated that the
MAPK pathway can either mediate or inhibit cell prolifera-
tion, depending on the induction of down-stream events,
most notably COX-2 meditated production of PGE
PGE has been demonstrated to be a potent inhibitor of
broblast (Mcanulty et al., 1997) and airway smooth muscle
2
. Indeed,
2
®
proliferation (Belvisi et al., 1998). This is even more pertinent
since OSM induces COX-2 expression in vascular endothelial
cells (Pourteau et al., 1999; Bernard et al., 1999). However, in
the current study, incubation of ®broblasts with the selective
COX-2 inhibitor nimesulide or the non-selective inhibitor
indomethacin did not in¯uence OSM-induced proliferation,
suggesting that the mitogenic e€ect of OSM was not
2
mediated by COX activation and PGE production. This
was con®rmed by Western analysis of COX-1 and COX-2
expression and by measurement of PGE2 release following
OSM exposure.
OSM is also a potent regulator of IL-6 release from a
variety of cell types (Brown et al., 1991). In turn, IL-6 has
been shown to be a mitogen for human lung ®broblasts
Figure 6 E€ect of OSM on human lung ®broblast pro-collagen
production. Fibroblasts were grown to con¯uence and incubated with
71
1
0 or 100 ng ml
measured by HPLC. Data are means of six independent experiments.
P50.05 compared to cells in serum free medium.
OSM for 48 h and the production of hyp
*
(
Roth et al., 1995). However, the results of this study clearly
show that OSM does not induce IL-6 production in
broblasts, suggesting that ®broblast production of IL-6 is
®
lung ®broblasts. In the current study we have identi®ed
speci®c OSMR on the cell surface by ¯ow cytometry and
immunohistochemistry.
not directly involved is OSM-induced proliferation of lung
®broblasts.
Alterations in the proliferation and activation of
®broblasts may in¯uence the progression of wound
healing. As such, an increase in viable ®broblast numbers
through inhibition of apoptosis may enhance the ®brotic
response. However, the role of OSM or related cytokines
in apoptosis has received little attention. The results of the
current study support this hypothesis since OSM inhibited
the development of spontaneous apoptosis in human lung
®broblasts over a 48 h period. We have previously shown
that while LIF has no e€ect on either spontaneous or
Fas-induced eosinophil apoptosis, OSM signi®cantly in-
hibits spontaneous apoptosis in these cells (Zheng et al.,
1999). OSM has also been shown to inhibit osteoblast
apoptosis by preventing an increase in the expression of
the pro-apoptotic protein Bax (Jilka et al., 1998). Taken
together, these data suggest that OSM may be anti-
apoptotic.
Further evidence of a pro®brotic role for OSM is its ability
to increase collagen in mesenchymal cells (Duncan et al.,
1995; Ihn et al., 1997; Bamber et al., 1998, Levy et al., 2000)
although the mechanisms involved may di€er between cell
types. Duncan et al. (1995) demonstrated an increase in both
types I and III procollagens and their mRNA transcripts in
human dermal ®broblasts and more recently a response
element has been characterized in this cell type that mediates
OSM stimulation of the human a2(I) collagen promoter (Ihn
et al., 1997). OSM also increased collagen protein levels in
human hepatic stellate cells (Levy et al., 2000) although an
increase in collagen a2(I) mRNA was not detected. As OSM
stimulated TIMP-1 production in these cells it was suggested
that TIMP-1 was responsible for or contributed to the
increased collagen protein by the inhibition of collagen
degradation. In the current study OSM also stimulated
collagen production in human lung ®broblasts although the
increase observed was only moderate when compared to the
TGF-b control. The mechanism for this increase is currently
being investigated.
Proliferation of human lung ®broblasts in response to
OSM were completely abrogated by speci®c antibodies
against OSMR and gp130, which con®rmed that OSM
preferentially engages and activates its speci®c type II
receptor. The maximal e€ect observed was at a concentration
71
of 2 ng ml . This mitogenic e€ect is consistent with a recent
study, which showed that OSM induced the proliferation of
dermal ®broblasts (Ihn & Tamaki, 2000).
The signal transduction pathways utilized by OSM have
traditionally been thought to involve tyrosine phosphoryla-
tion of multiple proteins including the Janus kinase (JAK)-
STAT pathway (Heinrich et al., 1998). To examine the role of
the tyrosine kinase activation in OSM-induced mitogenesis,
we treated ®broblasts with the speci®c tyrosine kinase
inhibitor genestein. In keeping with previous studies (Li &
Zafarullah, 1998; Heinrich et al., 1998), our results demon-
strate that OSM-induced mitogenesis of these cells was
completely inhibited by genestein. Phosphorylation of
tyrosine residues on the OSMR also provides binding sites
for a variety of signalling molecules containing SH2 domains,
including those that activate the ras-MAPK pathway. In
mammalian cells, three distinct MAPK families exist, p42/p44
MAPK, p38 MAPK and JNK (Gutkind, 1998). However, it
is the p42/p44 MAPK that plays a pivotal role in stimulating
®
broblasts to re-enter the cell cycle (Pages et al., 1993).
Activation of p42/p44 MAPK occurs through phosphoryla-
tion of threonine and tyrosine residues under the control of
the enzyme MAP kinase kinase (MEK) 1/2. In this study we
used the MEK-1/2 inhibitor PD98059, which prevents down-
stream activation of p42/p44 MAPK, but does not inhibit
p38 MAPK or JNK. Incubation of ®broblasts with PD98059
markedly diminished OSM-induced mitogenesis, strongly
suggesting that OSM stimulates the proliferation of ®bro-
blasts via a MAPK-dependent pathway. Similar ®ndings have
recently been reported for dermal ®broblasts (Ihn & Tamaki,
2
000).
British Journal of Pharmacology vol 136 (5)

800
A.K. Scaffidi et al
OSM exerts multiple effects on fibroblast function
In summary we have demonstrated that OSM has a
play a role in a variety of pathological conditions leading to
excessive collagen deposition such as pulmonary ®brosis and
asthma.
number of e€ects on ®broblast behaviour, including mitogen-
esis, inhibition of apoptosis and the capacity to stimulate
collagen deposition. These actions of OSM suggest an
involvement in limiting the tissue damage of an in¯ammatory
response as well as regulating tissue repair. Coupled with
previously demonstrated e€ects on TIMP-1, plasminogen
activator and acute phase proteins and the inhibition of pro-
in¯ammatory cytokines these data suggest that OSM may
play an important role in normal wound healing. However,
dysregulated production of OSM or its receptors may also
The authors would like to acknowledge the ®nancial support of the
National Health and Medical Research Council of Australia and
the Asthma Foundation of Western Australia. We also thank
Immunex Corp. for the provision of the OSMR antibody and Dr.
Neil Misso for his review of the manuscript. Yuben Moodley is the
inaugural recipient of the South African Pulmonology Society
Post-doctoral Research Fellowship to Australia and New Zealand.
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(
Received March 11, 2002
Revised April 4, 2002
Accepted April 18, 2002)
British Journal of Pharmacology vol 136 (5)