Relationship of Fiber Surface Iron and Active Oxygen Species to
Expression of Procollagen, PDGF-A, and TGF-␤1 in Tracheal Explants
Exposed to Amosite Asbestos
Jin Dai and Andrew Churg
Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada
To investigate the role of iron and active oxygen species
(AOS) in asbestos-induced fibrosis, we loaded increasing
amounts of Fe(II)/Fe(III) onto the surface of amosite asbestos
fibers and then applied the fibers to rat tracheal explants. Ex-
plants were harvested after 7 d in air organ culture. Asbestos
by itself doubled procollagen gene expression, and a further
increase was seen with increasing iron loading; actual col-
lagen content measured as hydroxyproline was increased in a
similar pattern. Iron loading also increased gene expression of
platelet-derived growth factor (PDGF)-A and transforming
growth factor (TGF)-␤1. Neither asbestos alone nor iron-loaded
asbestos affected gene expression of PDGF-B, tumor necrosis
factor-␣, or TGF-␣. The AOS scavenger tetramethylthiourea or
treatment of fibers with the iron chelator deferoxamine pre-
vented asbestos-induced increases in procollagen, PDGF-A, and
TGF-␤ gene expression, whereas glutathione had no effect.
The proteasome inhibitor MG-132 abolished asbestos-induced
increases in procollagen gene expression but did not affect in-
creases in PDGF-A or TGF-␤1 expression, whereas the extracel-
lular signal-regulated protein kinase (ERK) inhibitor PD98059
had exactly the opposite effect. We conclude that surface iron
as well as the iron-catalyzed generation of AOS play a role in
asbestos-induced matrix (procollagen) production and that
this process is driven in part through oxidant-induced nuclear
factor ␬B activation. Surface iron and AOS also play a role in
PDGF-A and TGF-␤ gene expression, but through an ERK-de-
pendent mechanism.
There is considerable evidence that active oxygen species
(AOS) are important mediators of asbestos toxicity (1–3).
Asbestos fibers themselves catalyze the formation of AOS
in aqueous media: Fe(II) on the surface of the fiber re-
duces molecular oxygen to superoxide anion, which then
dissociates to hydrogen peroxide, and hydrogen peroxide
reacts with Fe(II) to yield the highly reactive hydroxyl rad-
ical. Whether the catalytic iron is that actually on the fiber
surface or is iron that is mobilized from asbestos into the
medium is as yet not established, but it is clear that iron is
crucial to the process because chelation of fiber iron with
(Received in original form April 20, 2000 and in revised form September
30, 2000)
Address correspondence to: Andrew Churg, M.D., Dept. of Pathology,
University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC,
V6T 2B5 Canada. E-mail: achurg@interchange.ubc.ca
Abbreviations: xxx, AEBSF; active oxygen species, AOS; complementary
DNA, cDNA; deferoxamine, DFX; Dulbecco’s modified Eagle’s medium,
DMEM; ethylenediaminetetraacetic acid, EDTA; enzyme-linked immu-
nosorbent assay, ELISA; extracellular signal-regulated protein kinase, ERK;
glutathione, GSH; N-2-hydroxyethylpiperazine-N⬘-ethane sulfonic acid,
Hepes; hydroxyproline, HP; interleukin, IL; malate dehydrogenase, MDH:
nuclear factor, NF; platelet-derived growth factor, PDGF; phosphorylated
ERK, phosphoERK; reverse transcriptase polymerase chain reaction, RT-
PCR; transforming growth factor, TGF; tetramethylthiourea, TMTU; tu-
mor necrosis factor, TNF.
Am. J. Respir. Cell Mol. Biol. Vol. 24, pp. 427–435, 2001
Internet address: www.atsjournals.org
agents such as deferoxamine (DFX) that render it redox
inactive prevents the generation of AOS, and conversely,
chelation with agents such as ethylenediaminetetraacetic
acid (EDTA) that mobilize iron from the surface but leave
it redox active increases AOS generation (3).
Asbestos-induced AOS are believed to produce a vari-
ety of acute abnormalities, including lipid peroxidation,
protein oxidation, damage to DNA, activation of cell sig-
naling cascades, particularly nuclear factor (NF)-␬B, NF-
interleukin (IL)-6, and activator protein-1, and release of
acute inflammatory mediators such as IL-1, IL-8 (or the
murine macrophage inflammatory peptide-2), and tumor
necrosis factor (TNF)-␣ (1–5). AOS also increase adhesion
of fibers to the surface of epithelial cells (6) and increase
fiber uptake by epithelial cells (7), events that presumably
lead to increases in the generation of the mediators just listed.
Many of these effects can be prevented with DFX, again
implicating fiber surface iron as a fundamental mediator.
AOS may also be responsible for asbestos-induced auto-
phosphorylation of the epidermal growth factor (EGF) re-
ceptor and subsequent activation of the extracellular sig-
nal-regulated protein kinase (ERK) pathway (2, 8).
The role of AOS in chronic forms of asbestos toxicity is
much less well defined and is somewhat controversial.
Mossman and coworkers (9) showed that administration
of polyethylene glycol–conjugated catalase decreased in-
flammation and fibrosis in a rodent inhalation model of
early asbestosis, implying that rapid removal of hydrogen
peroxide, and/or prevention of formation of hydroxyl radi-
cal, was protective. Kamp and colleagues (10) reported
that the iron chelator phytic acid had a similar effect in an
instillation model, again implicating iron and AOS in the
development of fibrosis. However, the mechanisms by
which AOS might produce fibrosis are poorly defined, and
other data suggest that fibrogenic mediators are more im-
portant than AOS in fibrogenesis. Platelet-derived growth
factor (PDGF), transforming growth factor (TGF)-␤, and
TGF-␣ are all profibrotic peptides that cause fibroblast as
well as epithelial proliferation and increased matrix pro-
duction. Liu and associates (11, 12) and Perdue and Brody
(13) have shown, using a high concentration–brief expo-
sure inhalation model, that chrysotile asbestos exposure
upregulates macrophage and/or epithelial production of
PDGF, TGF-␤, and TGF-␣ as well as TNF-␣ and that in-
creased TNF-␣ production is crucial to the induction of
both mediators and fibrosis because mice with both TNF-␣
receptors knocked out are protected against the fibrogenic
effects of asbestos (14). However, the relationship of fiber-
induced AOS generation to the induction of fibrogenic
mediators is as yet uncertain, and it is also uncertain which
mediator(s) are most important in fibrogenesis.
428 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 24 2001
Examination of fibrogenic events in whole animal mod-
els is complicated by the presence of both inflammatory
and tissue reactions, and their interactions. We have de-
veloped a tracheal explant model that is free of exogenous
inflammatory cells and thus allows a simpler investigation
of fibrogenic processes in tissue. Using this model, we
have previously shown that exposure of explants to asbes-
tos results in increases in gene expression for PDGF-A,
TGF-␤, and procollagen, as well increases in actual col-
lagen content over a period of 7 d (15). In this study we
use the explant model to examine the effects of iron and
AOS on the production of fibrogenic mediators and matrix.
Materials and Methods
Dusts and Iron Loading
The asbestos used was the International Union Against Cancer
amosite standard reference sample. The geometric mean fiber size ⫾
standard deviation, as determined by counting by electron micros-
copy in our laboratory, was 3.8 ⫾ 2.7 ␮m in length and 0.26 ⫾ 1.9 ␮m
in width. To load iron onto the fiber surface, the fibers were treated
overnight with various concentrations of a freshly prepared mixture
of equimolar Fe(II)/Fe(III) chloride. The fibers were then washed
three times with saline to remove unbound iron and resuspended in
culture medium. We have previously shown that this procedure re-
sults in reproducible increases in measurable surface iron (6).
Explant Preparation and Culture
Tracheal explants were prepared from 250 g male Sprague-Daw-
ley mice as previously described (15). Each explant was approxi-
mately 2 ⫻ 2 mm. Because most of the explant by weight is carti-
lage and the amount of tissue that actually contributes RNA is
extremely small, three explants were used to prepare RNA for
each data point for reverse transcriptase polymerase chain reac-
tion (RT-PCR) analysis. We have previously shown that this pro-
cedure provides a reliable signal (15). Freshly prepared explants
from several different animals were used for each experiment
and mixed to ensure that all explants for a given data point did
not come from the same animal. Each test group consisted of
three data points.
For dust exposure, the explants were submerged, epithelial
side up, in a 500-␮g/cm2 (see DISCUSSION) suspension of dust in
Dulbecco’s modified Eagle’s medium (DMEM) without serum
for 1 h. Controls were exposed only to culture medium. At the
end of this time, the explants were transferred to petri dishes con-
taining DMEM in agarose supplemented with 1% glutamine, 1%
penicillin/streptomycin/fungizone, 1 ␮g/ml insulin, 0.1 ␮g/ml hy-
drocortisone, 1.5⫻ amino acids, and 10% chicken serum. Ex-
plants were maintained in air plus 5% CO 2 organ culture with
basal feeding in an incubator at 37 ⬚C for 7 d because previous in-
vestigation has shown that there are few changes in the level of
gene expression in this model before 7 d (15). All experiments
were repeated. Representative data from single experiments are
shown.
Measurement of Hydroxyproline
Hydroxyproline (HP) was measured on individual explants by
high performance liquid chromatography using the methodology
described previously (15).
Measurement of Phosphorylated ERK Protein Levels
Levels of phosphorylated ERK (phosphoERK) in the explants
were very low and six segments were combined to produce each
data point. The ERK inhibitor PD98059 was added in the same
fashion described in subsequent text to show specificity. To assay
phosphoERK levels, tracheal segments were frozen with liquid
nitrogen and ground to a fine powder. The pulverized tissue was
resuspended in lysing solution (20 mM Tris buffer with 1% Tri-
ton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA,
25 mM glycerophosphate, 2 mM pyrophosphate, 0.5 mM AEBSF,
10 ␮g/ml leupeptin, 20 ␮g/ml aprotinin, 1 mM orthovanadate,
and 10 mM sodium fluoride; all reagents from Sigma, St. Louis,
MO). The lysates were centrifuged at 14,000 rpm for 10 min at
4⬚C. Phospho-p44/42 mitogen-activated protein kinase was im-
munoprecipitated by using a specific anti-p44/42 antibody (Cell
Signaling, Beverly, MA) and protein A agarose beads (GIBCO-
BRL, Grand Island, NY). Proteins were resolved on a 15% poly-
acrylamide gel under reducing conditions and immobilized onto a
nitrocellulose membrane. For immunodetection, anti-p44/42 anti-
body was used with horseradish peroxidase–labeled secondary
antibody and visualized with enhanced chemiluminescence (Am-
ersham, Arlington Heights, IL). As a positive and negative con-
trol, phosphorylated and nonphosphorylated ERK proteins were
used (Cell Signaling).
Measurement of TGF-␤1 Protein Levels
Measurement was carried out using a Quantikine enzyme-linked
immunosorbent assay (ELISA) kit from R&D Systems (Minne-
apolis, MN). Because the explants are maintained in air organ
culture, explant tissue itself was used in the analysis. Explants
were homogenized under liquid nitrogen, and the powder was re-
suspended in 500 ␮l lysing solution on ice for 20 min. The lysing
solution contained 10 mM N-2-hydroxyethylpiperazine-N⬘-
ethane sulfonic acid (Hepes), pH 7.5, 0.5% Triton X-100, 150 mM
NaCl, 1 mM EDTA, 0.5 mM AEBSF, 1 ␮g/ml leupeptin, 1 ␮g/ml
aprotinin, 10 ␮g/ml trypsin-chymotrypsin inhibitor, and 1 ␮g/ml
pepstatin A (all reagents from Sigma). The samples were then
centrifuged at 14,000 rpm for 10 min at 4 ⬚C, and the supernatants
were decanted and kept on ice. TGF- ␤ was activated by adding
500 ␮l 2.5 N acetic acid/10 M urea solution to 500 ␮l supernatant.
This mixture was incubated at room temperature for 10 min and
then neutralized with 2.7 N NaOH/1 M Hepes. ELISA was then
performed according to the manufacturer’s instructions.
Effects of Added Iron Without Fibers
To determine whether the effects observed with iron loading
were specifically related to fiber-bound iron, explants without
dust were incubated with 1 mM Fe(II)/Fe(III) mixture described
previously for 1 h, then transferred to agarose culture dishes with
the same iron mixture added to the medium, and assayed after 7 d
in culture.
Treatment with Inhibitors and Chelators
Iron chelator DFX. Amosite asbestos was incubated overnight
with 10 mM DFX (Desferal; Ciba-Geigy) and excess DFX was
removed by washing the fibers in saline before use. Fibers were
then resuspended in culture medium and used as described previ-
ously.
Proteasome inhibitor MG-132. Explants were submerged in
0.5 ␮M MG-132 (Peptide Institute Inc., Osaka, Japan) in 0.1%
dimethyl sulfoxide/DMEM for 1 h and were then exposed to as-
bestos fibers as described previously, but with MG-132 in the me-
dium. A total of 0.5 ␮M MG-132 was also included in the agarose
culture medium for the 7-d incubation period.
AOS scavenger tetramethylthiourea . Explants were exposed
for 1 h to 10 mM tetramethylthiourea (TMTU) (Sigma) and then
to asbestos fibers with TMTU in the medium. A total of 10 mM
TMTU was also included in the agarose culture medium for 7 d.
ERK inhibitor PD98059 . Explants were exposed for 1 h to
50 ␮M PD98059 (Calbiochem, La jolla, CA) and then to asbestos
Dai and Churg: Fiber Surface Iron, Oxidants, and Fibrogenesis 429

fibers with PD98059 in the medium. A total of 50 ␮M PD98059 TABLE 1

was also included in the agarose culture medium for 7 d. Primer sequences for RT-PCR
Nonmembrane permeable AOS scavenger glutathione . Ex-
Target
plants were exposed for 1 h to 10 mM glutathione (GSH) (Sigma) Target Gene Size
and then to asbestos with GSH in the medium. GSH was also in- (Gen Bank) Primer Sequences (5⬘–3⬘) (bp)
cluded in the agarose medium for 7 d.
Procollagen F: cca atc tgg ttc cct ccc ac 181
Gel Shift Assay for NF-␬B (M27208) R: gta agg ttg aat gca ctt
TNF-␣ F: cat gat ccg aga tgt gga act ggc 315
To confirm that asbestos did activate NF- ␬B in our explant sys- (X66539) R: ctg gct cag cca ctc cag c
tem, additional explants were treated with asbestos or asbestos TGF-␣ F: gaa gca ggc cat cac tgc cct g 472
plus MG-132 as described previously and incubated for 7 d. Ex- (M31076) R: gtg ggt tag caa gaa ggc cag c
plants were snap-frozen and then homogenized in 0.1% Triton TGF-␤ F: cga ggt gac ctg ggc acc atc cat gac 404
X-100, 150 mM NaCl, 10 mM Hepes, pH 7.5, 1 mM EDTA, 0.5 (X52498) R: ctg ctc cac ctt ggg ctt gcg acc cac
mM AEBSF, 1 mg/ml leupeptin, 1 ␮g/ml aprotinin, 10 ␮g/ml soy- PDGF-A F: ctc ggc tgc gga tac ctc gc 403
bean trypsin inhibitor, and 1 ␮g/ml pepstatin A. The homogenate (Z14120) R: ctt gag ggc tgg cac ttg acg c
was incubated on ice for 5 min and then centrifuged at 5,000 rpm PDGF-B F: gct cct ttg atg acc ttc agc 282
for 5 min. The pelleted nuclei were resuspended in 100 to 500 ␮l (Z14117) R: cag ccc gag cag cgc tgc acc tc
of a solution of 25% glycerol, 20 mM Hepes, 420 mM NaCl, 1.2 Malate dehydrogenase F: caa gaa gca tgg cgt ata caa 507
mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.5 MDH (16) R: ttt cag ctc agg gat ggc ctc
mM AEBSF, 1 ␮g/ml leupeptin, 1 ␮g/ml aprotinin, 10 ␮g/ml soy-
bean trypsin inhibitor, and 1 ␮g/ml pepstatin A, and left on ice Definition of abbreviations: forward, F; reverse, R.
for a 30-min high salt extraction of the nuclear proteins. The lysed
nuclei were centrifuged at 5,000 rpm for 15 s and a protein assay
was carried on the supernatant. Single stranded NF- ␬B consensus For TNF-␣, expression was also examined at 2 and 8 h after
oligonucleotide (5⬘ - AGT TGA GGG GAC TTT CCC AGG C- 3⬘) dust exposure to determine whether the apparent lack of long-
was random labeled with [32P]cytidine triphosphate. Binding re- term upregulation might be hiding early upregulation. As a posi-
actions containing equal amounts of protein (7 ␮g) and 6.7 pmol tive control, explants were treated for 2 h with 20 ng/ml of recom-
of oligonucleotide were performed for 20 min in binding buffer binant human TNF-␣ (specific activity ⬎ 2 ⫻ 107 U/mg; Life
(10 mM Tris HCl, 50 mM NaCl, 1 mM EDTA, 4% glycerol, 67 Technologies, Rockville, MD).
␮g/ml poly(dI-dC)). Reaction products were separated in a 5%
polyacrylamide gel in 0.25⫻ TBE buffer and analyzed by autora- Statistics
diography and densitometry. Comparisons for gene expression and HP content were made
among treatment groups by analysis of variance.
Expression of Growth Factors, Fibrogenic Mediators,
and Matrix Components by RT-PCR Results
After 7 d in organ culture, explants were harvested and RNA ex- Analysis of type I procollagen gene expression and tissue
tracted by the method of Chomczynski and Sacchi (16). First HP levels were used as the measure of asbestos/iron ef-
strand complementary DNA (cDNA) was synthesized using super-
fects on matrix production. The effect of iron loading on
script RNase H reverse transcriptase (GIBCO-BRL) according
to the manufacturer’s instruction. Briefly, 5 ␮g RNA were added
procollagen gene expression is shown in Figure 1. Asbes-
to a reaction mixture of 1⫻ first strand buffer, 200 ng oligo(dT)12- tos by itself roughly doubled procollagen gene expression
18 primer, 0.5 mM each deoxyadenosine triphosphate, deoxythy- and there was a further progressive increase with increas-
midine triphosphate, deoxyguanidine triphosphate, and deoxycyti- ing levels of surface iron. Data on tissue HP levels are
dine triphosphate, 0.1 M DTT, plus water to 49 ␮l. A total of 200 shown in Figure 2: levels were significantly greater after
U superscript RT was added and the reaction incubated at 42 ⬚C asbestos treatment compared with control levels, and
for 1 h. greater again with loading with 1 mM iron (use of multiple
PCRs contained 1 ␮M primers, 1.5 mM Mg⫹⫹, 200 ␮M deoxy- iron loading levels was not attempted because the differ-
nucleotide triphosphates, reaction buffer, 2.5 U of Taq DNA ences in HP content are small). Figure 2 also shows that in-
polymerase (Perkin-Elmer Cetus Instruments, Norwalk, CT) and
cubation of control explants with 1 mM iron as described
1 or 5 ␮l of cDNA in a final volume of 20 ␮l. The PCR tempera-
ture profile consisted of 25 or 28 cycles of denaturation at 94 ⬚C
in MATERIALS AND METHODS, but without dust, did not in-
for 45 s, primer annealing at 60⬚C for 45 s, and extension at 72⬚C crease HP content. The effects of the iron chelator DFX
for 1.25 min, followed by an additional 5 min of final extension at and the cell permeable AOS scavenger TMTU, both of
72⬚C. The PCR products were size-fractionated on 1.5% agarose which completely abolished the asbestos-induced increases
gel and quantified from this ethidium bromide–stained gel using in procollagen gene expression, and the cell membrane im-
a gel documentation system (Bio-Rad Laboratories, Hercules, permeable AOS scavenger GSH, which did not prevent in-
CA). creased procollagen gene expression, are shown in Figure
Primer design was based on sequences from the Genbank da- 3. The effects of proteasome inhibitor MG-132 (used here
tabase (Table 1). We optimized the reaction conditions (magne- as an inhibitor of NF-␬B activation) and the ERK inhibi-
sium concentration, thermocycler temperature, etc.) to produce
tor PD98059 are shown in Figure 4; the former completely
the greatest amount of a single PCR product. The amount of
cDNA used and number of cycles of amplification were adjusted
prevented the asbestos-induced increases, whereas PD98059
to stay within the linear region of amplification in order to allow did not affect asbestos-mediated increases in procollagen
quantification. Specificity of the various products was confirmed gene expression at all.
by restriction digests. Expression of malate dehydrogenase (17) To show that asbestos did activate NF-␬B in our ex-
was used as control (housekeeper) gene. plant system, a gel shift assay was run and is shown in Fig-
430 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 24 2001
Figure 1. Increasing levels of iron loading on amosite asbestos in-
crease procollagen gene expression. Ethidium bromide images
show RT-PCR products, with densitometry below. MDH ⫽
housekeeper (malate dehydrogenase). A only ⫽ asbestos alone;
A/[x] mM Fe ⫽ asbestos ⫹ [concentration] of loaded iron. Val-
ues are mean ⫾ SD. *Significantly greater than control. Asbes-
tos/0.1 mM Fe is also significantly greater than asbestos alone,
and asbestos/1 mM Fe is greater than asbestos/0.1 mM Fe.
ure 5. The gel shift assay is carried out on nuclear extracts
and indicates the relative amount of NF-␬B protein in the
nucleus (i.e., translocated NF-␬B). Asbestos increased
NF-␬B activity and this increase was prevented by inclu-
Figure 2. Tissue hydroxyproline content is increased in asbestos-
treated explants compared with control explants and further in-
creased by loading with 1 mM iron. These findings indicate that
hydroxyproline production is a function of fiber surface iron lev-
els. Addition of iron to the medium (“iron alone”) without dust
has no effect on hydroxyproline content. Values are mean ⫾ SD.
*Significantly greater than control. Asbestos ⫹ 1 mM Fe is also
significantly greater than asbestos alone.
Figure 3. The iron chelator DFX and the membrane permeable
AOS scavenger TMTU protect against asbestos-induced in-
creases in procollagen gene expression, indicating that the iron-
catalyzed generation of AOS plays a role in increased procol-
lagen expression. The nonmembrane permeable AOS scavenger
GSH is not protective, indicating that the important reactions must
be intracellular. A Only ⫽ asbestos alone. Values are mean ⫾
SD. *Significantly greater than control.
sion of MG-132 in the medium. To show that asbestos fi-
bers did activate the ERK pathway, proteins were ex-
tracted and phosphoERK levels were measured on Western
blot. As shown in Figure 6, phosphoERK levels were greater
Figure 4. Proteasome inhibitor MG-132 prevents asbestos-induced
increases in procollagen expression, whereas ERK inhibitor
PD98059 does not, suggesting that NF-␬B activation is involved
in increased procollagen expression. Values are mean ⫾ SD. A
only ⫽ asbestos alone. *Significantly greater than control.
Dai and Churg: Fiber Surface Iron, Oxidants, and Fibrogenesis
in asbestos-exposed explants compared with control explants
and greater again in explants treated with iron-loaded as-
bestos. The ERK inhibitor PD98059 completely abolished
asbestos-induced increases in phosphoERK.
Figures 7 to 9 show similar data for TGF-␤1 gene ex-
pression and Figures 10 to 12 for PDGF-A gene expres-
sion. Asbestos by itself increased expression of both medi-
ators, and adding surface iron produced further increases.
DFX and TMTU again abolished the asbestos effect, but
GSH did not. In contrast to the situation for procollagen,
MG-132 did not prevent asbestos-induced increases in
Figure 6. Treatment with asbestos increases the level of phos-
phoERK, and treatment with asbestos ⫹ 1 mM iron increases it
still further. Addition of the ERK inhibitor PD98059 to the me-
dium prevents asbestos-induced increases in phosphoERK. Data
shown are representative Western blots with each signal derived
from six explants combined. 1 ⫽ control; 2 ⫽ asbestos alone; 3 ⫽
asbestos ⫹ 1 mM Fe iron; 4 ⫽ asbestos ⫹ PD989059 as described
in text; 5 ⫽ negative control (nonphosphorylated ERK); 6 ⫽ pos-
itive control (phosphoERK).
431
Figure 5. Gel shift assay showing that asbes-
tos exposure increases NF-␬B activation in
explants after 7 d of culture. Inclusion of
MG-132 in the medium prevents this effect.
1 ⫽ control; 2 ⫽ asbestos; 3 ⫽ asbestos ⫹ MG-
132. Each bar represents data from six ex-
plants combined.
PDGF-A or TGF-␤ gene expression, but PD98059 did.
Attempts to examine levels of TGF-␤ protein by ELISA
were unsuccessful: tissue levels were at the bottom of the
ELISA range and showed no differences among treatment
groups (data not shown). It is unclear whether there really
are no differences in TGF-␤ content (i.e., increased gene
expression is not translated into protein) or the protein
levels are simply too low to detect; however, in some
senses this question is not crucial because it is clear that
blocking PDGF or TGF-␤ gene expression with PD98059
does not prevent increases in procollagen gene expression,
thus PDGF and TGF-␤ are not driving collagen produc-
tion in this system.
The effects of asbestos and iron loading on TNF-␣ ex-
pression are shown in Figure 13: neither asbestos nor iron-
loaded asbestos increased expression. A similar lack of ef-
fect was seen in explants incubated for only 2 or 8 h (data
not shown). Exogenous TNF-␣, used as a positive control,
did upregulate explant TNF-␣ levels within 2 h (Figure
13). Inclusion of AOS scavengers, MG-132, and PD98059
did not affect expression of TNF-␣ (data not shown). Iden-
tical results were seen for PDGF-B and TGF-␣ expression
(data not shown).
Discussion
We have used a tracheal explant system to examine the role
of AOS and surface iron on the expression of asbestos-
induced fibrogenic mediators and matrix. As noted previ-
ously, examining fibrogenic events can be difficult, and the
explants simplify this process because they lack inflamma-
tory cells but preserve the important modulating influ-
ences of epithelial cells on mesenchymal cells and vice
versa (18). However, the explant system also has its pecu-
liarities: dust uptake is very slow and gene expression ap-
pears to follow dust uptake, hence 7-d cultures are re-
quired to see effects (15). As well, relatively high dust
loadings are required to produce effects. However, the
dose that the epithelium “sees” is probably very much
lower because we have shown in experiments looking at fi-
ber adhesion that the vast majority of fibers on the explant
surface are only very loosely adherent (6). Nonetheless,
these limitations need to be kept in mind in interpreting
our results.
Our data suggest that AOS and surface iron play an
432 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 24 2001
Figure 7. Increasing levels of iron loading on amosite asbestos in-
crease TGF-␤1 gene expression. Values are mean ⫾ SD. A only ⫽
asbestos alone; A/[x] mM Fe ⫽ asbestos ⫹ [concentration] of
loaded iron. *Significantly greater than control. Asbestos/0.1 mM
Fe is also significantly greater than asbestos alone, and asbes-
tos/1 mM Fe is greater than asbestos/0.1 mM Fe.
important role in the development of asbestos-induced fi-
brosis and affect expression of both matrix proteins and fi-
brogenic mediators, but in different ways. The current ex-
periments show that it is a clear relationship between
increasing levels of surface iron and increases in the ex-
Figure 8. DFX and TMTU protect against asbestos-mediated in-
creases in TGF-␤ gene expression, whereas GSH does not. Values
are mean ⫾ SD. A only ⫽ asbestos alone. *Significantly greater
than control.
Figure 9. Proteasome inhibitor MG-132 does not prevent asbes-
tos-induced increases in TGF-␤ gene expression, whereas ERK
inhibitor PD98059 is protective. Values are mean ⫾ SD. A only ⫽
asbestos alone. *Significantly greater than control.
pression of procollagen messenger RNA (mRNA) and HP,
and that addition of a cell permeable AOS scavenger
(TMTU) or an iron chelator (DFX) that renders surface
iron redox inactive prevents asbestos-induced increases in
procollagen expression. These observations imply that
Figure 10. Increasing levels of iron loading on amosite asbestos
increase PDGF-A gene expression. Values are mean ⫾ SD. A
only ⫽ asbestos alone; A/[x] mM Fe ⫽ asbestos ⫹ [concentra-
tion] of loaded iron. *Significantly greater than control. Asbes-
tos/0.1 mM Fe is also significantly greater than asbestos alone,
and asbestos/1 mM Fe is greater than asbestos/0.1 mM Fe.
Dai and Churg: Fiber Surface Iron, Oxidants, and Fibrogenesis
Figure 11. DFX and TMTU protect against asbestos-mediated
increases in PDGF-A gene expression, whereas GSH does not.
Values are mean ⫾ SD. A only ⫽ asbestos alone. *Significantly
greater than control.
asbestos-induced increases in collagen production are
driven, at least in part, by AOS, presumably through the
iron-catalyzed formation of hydroxyl radical. Thus, these
observations support the previous findings of Mossman
and coworkers (9) and Kamp and colleagues (10) that sug-
gested that asbestos can induce fibrosis through an oxidant
Figure 12. Proteasome inhibitor MG-132 does not prevent asbes-
tos-induced increases in PDGF-A gene expression, whereas
ERK inhibitor PD98059 is protective. Values are mean ⫾ SD. A
only ⫽ asbestos alone. *Significantly greater than control.
433
Figure 13. Neither asbestos alone nor iron-loaded asbestos affect
gene expression of TNF-␣. GSH, DFX, and TMTU similarly had
no effect, and inclusion of MG-132 or PD98059 in the medium
also did not change expression levels (data not shown). Identical
results were seen for PDGF-B and TGF-␣. A only ⫽ asbestos
alone; A/[x] mM Fe ⫽ asbestos ⫹ [concentration] of loaded iron.
Minus and plus indicate a separate experiment done to show that
incubation of explants with 20 ng/ml of TNF-␣ for 2 h (⫹) leads
to upregulation of TNF-␣ compared with untreated explants (⫺).
Densitometry is not shown, but increase is about 2.5-fold.
mechanism. Because our explant system has no exogenous
inflammatory cells, it is clear that AOS derived from the
fibers are able, in and of themselves, to cause procollagen
expression. However, all dusts evoke an inflammatory re-
sponse, and it is possible that, in vivo, AOS derived from
dust-evoked inflammatory cells augment this process (see
subsequent text). Also of interest is the observation that
the nonmembrane permeable AOS scavenger GSH was
not protective, which implies that fiber-derived AOS must
be acting within epithelial and/or interstitial cells rather
than on the cell surface, an idea supported by our previous
observation that increases in gene expression parallel
(over time) increases in dust uptake by the epithelial and
interstitial cells (15).
There is evidence in other experimental systems that
iron affects collagen production. This effect has been seen
most clearly in hepatocytes, where iron loading of the sort
found in hemochromatosis has been shown to increase the
activity of prolyl hydroxylase, a crucial enzyme for col-
lagen production (19, 20). Iron overload also causes hepa-
tocytes to increase expression of type I procollagen (19,
20). In a model more relevant to this study, Gardi and as-
sociates (20) showed that mobilizing iron from crocidolite
asbestos with redox active chelators such as citrate in-
creased collagen production by cultured rat lung fibro-
blasts. Unfortunately, our data do not allow us to deter-
mine whether the crucial effects in our explants occur in
434 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 24 2001
epithelial or interstitial cells, but it is of interest that add-
ing iron to the culture medium did not affect generation of
procollagen, implying either that iron bound to the asbes-
tos fiber surface is crucial to this process or that iron must
be mobilized from the fiber surface with the appropriate
chelator to be active in fibrogenesis.
NF-␬B resides in the cytoplasm as an inactive DNA-
binding dimer (NF-␬B itself) and an inhibitory protein,
I␬B. NF-␬B can be activated by a variety of pathways,
most commonly when an external stimulus leads to I␬B
phosphorylation, ubiquitination, and consequent degrada-
tion in proteasomes. The NF-␬B complex then migrates to
the nucleus and activates a wide variety of genes involved
in the acute inflammatory response (21, 22). MG-132 is a
proteasome inhibitor that prevents degradation of I␬B
(23). As noted previously, asbestos fibers have been shown
to activate NF-␬B in monolayer tissue culture systems (1,
2), and we have confirmed with the gel shift assay that
NF-␬B is similarly activated over a relatively long period in
our explants. The fact that MG-132 completely prevents the
asbestos-induced increases in procollagen expression, not
only with the native fibers but also with the iron-loaded fi-
bers (data not shown), suggests strongly that increased
procollagen production is mediated in some fashion through
AOS-induced activation of NF-␬B. It is possible that pro-
lyl hydroxylase has an NF-␬B recognition site in its pro-
moter, but there appears to be no data on this question,
and several intermediate steps may also be involved.
Oxidant attack is known to activate NF-␬B (21, 24, 25),
and the present experiments thus support other observa-
tions that the generation of AOS from asbestos fibers di-
rectly activates NF-␬B (1, 2). However, what is surprising
is that there is no clear evidence for involvement of TNF-␣
in our model. The in vivo studies of Liu and coworkers (11,
12, 14) and Perdue and Brody (13) indicate that TNF-␣ is
crucial to chrysotile asbestos–induced fibrogenesis be-
cause mice lacking TNF-␣ receptors are protected. TNF-␣
itself activates NF-␬B (24, 25) and it is possible that, in
vivo, the production of TNF-␣ from asbestos-evoked mac-
rophages simply overshadows the direct effects of the dust,
particularly with chrysotile asbestos which has relatively
little iron compared with the amosite used here.
Explant tracheal epithelial cells certainly can upregu-
late TNF-␣ expression, as is clear from our positive (TNF-␣
driven) control. In this and our previous work (15), we
were unable to find evidence of upregulation of TNF-␣
gene expression from 2 h to 7 d of asbestos exposure, so it
is unlikely that we have missed a brief episode of increased
transcription followed by prolonged increases in protein
production. It is noteworthy that iron loading also had no
effect on TNF-␣ expression in our explants. Although it is
possible that increased levels of TNF-␣ do occur and are
produced purely by increased translation of pre-existing
mRNA or by increased release of preformed TNF-␣, this
appears unlikely because in other cell types, asbestos-
related stimuli to TNF-␣ production do increase gene ex-
pression. For example, Simeonova and Luster (26) have
shown that iron loading of asbestos increases TNF-␣ gene
expression in cultured alveolar macrophages by greater
than 10-fold.
Although we do not have a definite explanation of why
TNF-␣ gene expression is not upregulated in our explants,
the roles of AOS and TNF-␣ in activating NF-␬B are com-
plex. It is becoming clear that TNF-␣ and oxidants activate
NF-␬B via quite different mechanisms (21, 24, 27). This
applies not only to nuclear translocation but to generation
of transcriptionally competent NF-␬B bound to promotor
elements in DNA. Even oxidative stress itself appears to
activate separate pathways leading to NF-␬B DNA bind-
ing and NF-␬B transactivation (21). Whether and to what
degree these types of differences occur in tracheal epithe-
lial cells are not known but the important point is that
AOS derived from the dust itself and TNF-␣ derived from
airspace macrophages that have phagocytized dust may
activate different genes. Thus, it is possible that TNF-␣ de-
rived from airspace macrophages is required to upregulate
explant TNF-␣ and that AOS are not sufficient. This no-
tion might provide an explanation for the observation that
in the model developed by Liu and coworkers (11, 12, 14)
and Perdue and Brody (13), chrysotile asbestos upregu-
lates expression of TNF-␣, PDGF-B, and TGF-␣, none of
which is increased in our explants. Some indirect support
for this idea comes from the observation by Gallucci and
colleagues (28) that TNF-␣ upregulates expression of TGF-␣
in murine liver cells.
Relatively little information is available about the ef-
fects of AOS on induction of TGF-␤ and PDGF. Bellocq
and coworkers (29) recently reported that exposure of cul-
tured A549 cells (a model of human type II cells) to AOS
resulted in increased TGF-␤1 release, largely through in-
creased transcription. AOS are also known to activate la-
tent TGF-␤ (30). The current data support a role for iron-
derived AOS in TGF-␤1 transcription. It has also been
reported that high concentrations of GSH prevent release
of PDGF from platelets (31), possibly indicating that
PDGF production can be driven by AOS. Our data show
that this process differs for different forms of PDGF be-
cause in our hands both asbestos and iron-loaded asbestos
only upregulate gene expression of PDGF-A, a finding in
accord with the observations of Lasky and associates (32,
33) that chrysotile asbestos particularly stimulates produc-
tion of PDGF-AA and upregulates the PDGF-receptor ␣.
What is clear from our data is that in this model system,
AOS drive gene expression of PDGF-A and TGF-␤ in a
fashion quite different from that of procollagen because
only the ERK inhibitor PD98059, but not the NF-␬B in-
hibitor MG-132 prevents PDGF-A and TGF-␤1 upreg-
ulation and vice versa. As noted previously, Robledo
and Mossman (2) and Zanella and coworker (8, 34) have
shown that crocidolite asbestos activates the EGF recep-
tor, possibly via AOS, and that this leads, through an
ERK-dependent mechanism, to increases in c-fos expres-
sion and apoptosis. The same group has recently reported
that increased phosphoERK is detectable by immunohis-
tochemistry in the areas of beginning fibrosis in chrysotile-
exposed mice (35). Our data also suggest that asbestos-
induced ERK pathway activation is mediated, at least in
part, by AOS and that this pathway may be involved in as-
bestos- induced activation not only of genes involved in
acute responses but of a variety of genes involved in chronic
fibrotic responses. Thus, fibrogenesis, at least in our model,
appears to proceed via two separate pathways: one directly
Dai and Churg: Fiber Surface Iron, Oxidants, and Fibrogenesis
affects procollagen production and the other affects ex-
pression of fibrogenic mediators.
Acknowledgments: This study was supported by grant MA8051 from the Medi-
cal Research Council of Canada.
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