Induction of arginase I and II in bleomycin-induced fibrosis of mouse lung
Motoyoshi Endo,1,2
Seiichi Oyadomari,1
Yasuhiro Terasaki,3
Motohiro Takeya,3
Moritaka Suga,2
Masataka Mori,1 and
Tomomi Gotoh1
1Department of Molecular Genetics,
2First Department of Internal Medicine, and
3Second Department of Pathology, Kumamoto University
School of Medicine, Kumamoto 860-0811, Japan
Submitted 17 December 2002
; accepted in final form 31 March 2003
 |
ABSTRACT
|
---|
Arginase, which hydrolyzes arginine to urea and ornithine, is a precursor
for the synthesis of polyamines and proline, which is abundant in collagen.
The supply of proline can be a crucial factor in the process of lung fibrosis.
We investigated the induction of arginine metabolic enzymes in
bleomycin-induced mouse lung fibrosis. Histological studies and quantification
of lung hydroxyproline showed that lung fibrosis develops in up to 14 days
after bleomycin treatment. Under these conditions, collagen I mRNA was induced
gradually in up to 15 days, and the content of hydroxyproline reached a
maximum at 10 days. Arginase I mRNA was undetectable before bleomycin
treatment but was induced 510 days after this treatment. Arginase I
protein was induced at 7 days and remained little changed for up to 10 days
and decreased at 14 days. On the other hand, arginase II mRNA that was
detectable before treatment was increased gradually for up to 10 days and
decreased at 14 days. Arginase II protein began to increase at day 5,
increased for up to 10 days, and was decreased at day 14. mRNAs for
cationic amino acid transporter-2 and ornithine decarboxylase were induced in
a manner similar to that seen with collagen I mRNA. Immunohistochemical
analysis showed that arginase I is induced in macrophages, whereas arginase II
is induced in various cell types, including macrophages and myofibroblasts,
and roughly colocalizes with the collagen-specific chaperone heat shock
protein 47. Our findings suggest that arginine metabolic enzymes play an
important role in the development of lung fibrosis, at least in mice.
macrophage; lung fibrosis; arginine; collagen
IDIOPATHIC PULMONARY FIBROSIS (IPF), also known as cryptogenic
fibrosing alveolitis, is one of the groups of idiopathic pneumonia sharing the
clinical features of shortness of breath, radiographically evident diffuse
pulmonary infiltrates, and varying degrees of inflammation and fibrosis
(17). IPF is thought to result
from uncontrolled inflammatory processes that ensue from a variety of insults
to the lower respiratory tract of susceptible individuals
(17). However, the primary
insult of IPF is unknown. Fibrotic areas vary characteristically depending on
age and activity. Regions of chronic lung injury with scarring and cystic air
spaces contrast with regions of acute injury with foci of actively
proliferating fibroblasts and myofibroblasts. These focal zones of fibroblast
proliferation are located in sites of a recent alveolar injury. The exuberant
cellular response at these sites is similar to the healing processes observed
in skin and other tissues. The deposition of collagen takes place during the
healing process at the end stage of alveolitis. The primary structural motif
of mature collagen is (Gly-X-Pro/Hyp)n. Therefore, the supply of
proline, which is hydroxylated in the peptide to form hydroxyproline, can be a
limiting factor in the process of fibrosis.
Bleomycin is an anticancer agent prescribed for various cancers, including
that of the lung. However, this drug has a dose-dependent pulmonary toxicity,
including lung fibrosis, which limits its clinical use
(9). The toxic effect of this
agent has been utilized advantageously in a number of experimental approaches
to induce pulmonary fibrosis in animal models. Several groups of workers have
reported that in the early stages of bleomycin-induced lung damage, the
lesions are associated with biochemical and functional changes that resemble
those of human pulmonary fibrosis, including inflammatory cell infiltration,
increased collagen content, and reduced lung volume and compliance
(3,
5,
7,
37,
43,
44,
4749,
57).
Arginine, a precursor for the synthesis of urea, polyamines, creatine
phosphate, nitric oxide (NO), and proteins
(Fig. 1), derives from the cell
exterior by the cationic amino acid transporters (CATs) or is synthesized from
citrulline by successive actions of argininosuccinate synthetase (AS) and
argininosuccinate lyase (AL), the third and fourth enzymes of the urea cycle
(ornithine cycle). Ornithine, a product of arginase reaction, is converted to
glutamate and pyrrolidine-5-carboxylate by a mitochondrial enzyme, ornithine
aminotransferase (OAT). Pyrrolidine-5-carboxylate can be metabolized further
to proline, an amino acid abundant in collagen. Durante et al.
(11) reported that proline
generation was inhibited by the OAT inhibitor L-canaline, a finding
that means that OAT is important for the production of proline. Ornithine is
also a precursor for the synthesis of polyamines, which play an important role
in cell proliferation and growth
(22). The activity of
ornithine decarboxylase (ODC), the rate-limiting enzyme of the
ornithine-polyamine pathway, parallels intensity of the repair processes in
injured tissues (13).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1. Arginine metabolism. Arginine is a precursor for the synthesis of urea,
polyamines, nitric oxide (NO), and proteins including collagen. ODC, ornithine
decarboxylase; OAT, ornithine aminotransferase; P5C, pyrroline-5-carboxylic
acid; NOS, NO synthase; AS, argininosuccinate synthetase; AL,
argininosuccinate lyase; CAT, cationic amino acid transporter.
|
|
Arginase exists in two isoforms, the hepatic type (arginase I) and the
extrahepatic type (arginase II)
(32). Arginase I and II genes
were mapped to chromosomes 6q23 and 14q24, respectively. We reported that
CCAAT/enhancer binding protein family transcription factors bind to the
arginase I gene promoter and enhancer and play crucial roles in arginase I
expression (14,
24,
25). It was also reported that
Th1 cytokines suppress arginase I expression, whereas Th2 cytokines enhance it
(30). Regulation of arginase
II expression by cytokines was reported
(26,
50). We found that both
arginase I and II can prevent NO-dependent apoptosis in murine
macrophage-derived RAW 264.7 cells by depleting intracellular arginine and
thus decreasing NO production
(15). We also showed that
inducible nitric oxide synthase and arginase II are induced early in the lungs
of LPS-treated mice, whereas arginase I is induced late in the lung
(40). The induction of
arginase and involvement of several cytokines were also noted in some
inflammatory models (27,
45). Ornithine, which is
formed by arginase I and/or II in endotoxemia, may be utilized for synthesis
of polyamines and proline (and thus collagen), which are required for cell
growth and tissue repair. Ignarro et al.
(19) showed that ornithine,
which is formed by arginase, is utilized for polyamine synthesis in rat aortic
smooth muscle cells. They also reported that arginase activity is induced in
the process of collagen synthesis
(19). Durante et al.
(10) reported that the
physiological cyclic stretch directs arginine transport and metabolism to
collagen synthesis in vascular smooth muscle, and Shearer et al.
(41) noted induction of
arginase activity in wound healing. Therefore, it was deemed necessary to
determine whether arginase isoforms and other arginine metabolic enzymes are
involved in the process of lung fibrosis.
We now report that both arginase I and II are induced in the process of
bleomycin-induced fibrosis of mouse lung. We also found that arginase I is
induced in macrophages, whereas arginase II is induced in various cells,
including macrophages and myofibroblasts. mRNAs for CAT-2 and ODC were also
induced.
 |
MATERIALS AND METHODS
|
---|
Mice and treatment. Specific pathogen-free male C57BL/6 mice
weighing 1922 g (6 wk of age) were given 10 mg/kg of bleomycin
hydrochloride (Nippon Kayaku, Tokyo, Japan) intratracheally in 40 µl of
saline and the controls in the same volume of saline. All these mice were
killed at the indicated times following anesthetization with ether. All
procedures involving animals were approved by the Animal Care and Use
Committee of Kunamoto University.
RNA blot analysis. Total RNAs from mouse tissues were prepared by
the guanidium thiocyanate-phenol-chloroform extraction procedure
(8). After electrophoresis in
formaldehyde-containing agarose gels, the RNAs were transferred to nylon
membranes. For the hybridization, we used the following probes:
digoxigenin-labeled antisense RNAs for rat arginase I
(45), rat arginase II
(16), rat CAT-2
(40), rat ODC
(40), rat AS
(56), rat OAT
(38), and rat collagen type I.
The template plasmid for collagen I was prepared as follows. A partial cDNA
for rat collagen I corresponding to nucleotides 3,4153,882 (GenBank
accession number S67530
[GenBank]
) was isolated by using RT-PCR and rat liver RNA, then
inserted into the HincII site of pGEM-3Zf(+) (Promega, Madison, WI).
Chemiluminescence signals derived from the hybridized probes were detected
with a Las-1000 Plus bioimage analyzer (Fuji Photo Film, Tokyo, Japan) and the
digoxigenin luminescence detection kits (Roche Molecular Biochemicals,
Indianapolis, IN).
Immunoblot analysis. For immunoblot analysis of the cytosolic
proteins arginase I and heat shock cognate protein (Hsc) 70, the mouse lungs
were homogenized in 25 mM Tris · HCl (pH 7.4) containing 300 mM NaCl
and 1% Triton X-100, and the homogenates were centrifuged at 50,000 g
for 20 min at 4°C. The supernatants served as tissue extracts. For
analysis of arginase II and Tom20
(55), both of which are
mitochondrial proteins, the lungs were homogenized in 0.2 M KCl containing
0.25 M sucrose, and the homogenates were centrifuged at 650 g for 5
min at 4°C. The supernatants were centrifuged at 11,000 g for 20
min at 4°C. The pellet was dissolved in 25 mM Tris · HCl (pH 7.4)
containing 300 mM NaCl and 1% Triton X-100 and was used as mitochondrial
extracts that were subjected to SDS-PAGE. The proteins were electrotransferred
to nitrocellulose membranes. Immunodetection was done with ECL kits (Amersham,
Bucking-hamshire, UK) according to the protocol provided by the
manufacturer.
Immunohistochemical staining. Lungs were fixed with 4%
paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4), and the excised
tissues were embedded in optimum cutting temperature compound (Miles, Elkhart,
IN) and frozen in dry ice, and then the sections (5-µm thick) were cut and
air-dried. The digested sections were pretreated with 5 mM periodic acid for
10 min at room temperature to inhibit endogenous peroxidase activity. The
specimens were incubated for 1 h with 1,000-fold-diluted rabbit antiserum
against rat arginase I (20) or
rat arginase II (38) or mouse
monoclonal antibody F4/80
(28), which recognizes a
160-kDa glycoprotein on the cell surface of most mouse macrophage populations,
then were washed three times with PBS for 5 min. For the single immunostaining
of arginase I or II, the sections were incubated for 1 h with 500-fold-diluted
sheep anti-rabbit Ig[F(ab')2] conjugated with peroxidase
(Amersham) as a second antibody, and peroxidase activity was visualized by
incubation with a 3,3'-diaminobenzidine solution. For double staining of
arginase I or II and macrophages, sections were incubated for 1 h with Alexa
Fluor 488-labeled anti-mouse IgG (Molecular Probes, Eugene, OR) and Alexa
Fluor 546-labeled anti-rabbit IgG as second antibodies. For the double
staining of arginase II and heat shock protein (HSP) 47
(6), specimens were incubated
for 1 h with 200-fold-diluted antisera against rat arginase II and mouse
HSP47. The sections were then incubated for 1 h with Cy2-labeled anti-mouse
IgG (Amersham Pharmacia Biotech, UK) and Cy3-labeled anti-rabbit IgG as second
antibodies. For the double staining of arginase II and
-smooth muscle
actin, specimens were incubated for 1 h with 200-fold-diluted antisera against
rat arginase II and 500-fold-diluted mouse
-smooth muscle actin (Sigma
Chemical, St. Louis, MO). The sections were then incubated for 1 h with
Cy2-labeled anti-mouse IgG and Cy3-labeled anti-rabbit IgG as second
antibodies.
Hematoxylin-eosin and AZAN staining. Lungs were fixed by perfusing
with 4% paraformaldehyde in PBS (pH 7.4), embedded in paraffin, and sectioned
5-µm thick. The procedures of hematoxylin-eosin (HE) and AZAN staining were
as described (52).
Hydroxyproline quantification. To assess collagen contents in the
lung, we measured hydroxyproline content, as described
(54). In brief, a minced lung
was homogenized in 6 N HCl and hydrolyzed for 5 h at 130°C. The pH was
adjusted to 6.57.0 with NaOH, and the sample volume was adjusted to 30
ml with H2O. The sample solution (1.0 ml) was mixed with 1.0 ml of
chloramine T solution (0.05 M), and then the mixture was incubated at room
temperature for 20 min. When 1.0 ml of 20% dimethyl benzaldehyde solution was
added, the mixture was incubated at 60°C for 20 min. The absorbance at 560
nm was measured.
 |
RESULTS
|
---|
Development of lung fibrosis in bleomycin-treated mice. We induced
lung fibrosis in male C57BL/6 mice by giving bleomycin intratracheally. In
preliminary experiments, we titrated the dose of bleomycin, assessed the
degree of fibrosis by histochemical staining, and analyzed induction of mRNAs
for arginase I and II by Northern blot analysis (see Induction of arginase
I in macrophages and of arginase II in various cells in bleomycin-treated
mouse lungs). Maximal fibrosis and maximal induction of arginase I and II
mRNAs were obtained with 10 mg/kg of bleomycin hydrochloride, although about
one-third of mice died before 14 days. Thus bleomycin hydrochloride at 10
mg/kg was used in the following experiments. The development of lung fibrosis
was evaluated 14 days after this treatment
(Fig. 2). HE staining revealed
diffuse alveolar destruction with collapse and obliteration of alveolar
spaces, and collagen deposition was evident with AZAN staining. Collagen
contains hydroxyproline as a unique component. To evaluate collagen contents
in the lung, we measured hydroxyproline
(Fig. 3), which was
50
µg in control lungs. The hydroxyproline markedly increased 5 days after
treatment and reached a maximum (200 µg) at 10 days with little decrease at
14 days. Thus lung fibrosis is indeed induced by bleomycin with development of
lung fibrosis being completed in
10 days.

View larger version (125K):
[in this window]
[in a new window]
|
Fig. 2. Development of lung fibrosis in bleomycin-treated mice. Lungs of control
(A, C) and a bleomycin-treated mouse (14 days) (B, D) were
stained with hematoxylin-eosin (HE) (A, B) or AZAN (C, D).
Original magnification: x200.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3. Measurement of hydroxyproline of the bleomycin-treated mouse lung.
Bleomycin was given to mice intratracheally. After the indicated number of
days, hydroxyproline of the lungs was measured as described in MATERIALS
AND METHODS. Results are shown as means ± SD (n =
3).
|
|
Induction of arginine metabolic enzymes in the bleomycin-treated mouse
lung. We analyzed the induction of collagen I and arginine metabolic
enzymes in bleomycin-treated mouse lung by using RNA blot analysis
(Fig. 4). Collagen I mRNA was
present at a low level in untreated mice, began to increase at 3 days, and
increased gradually for up to 15 days, a finding in good agreement with data
in Fig. 3. Arginase I mRNA was
not detected in the control lung, was induced 5 days after bleomycin
treatment, reached a maximum at 7 days, and decreased to undetectable levels
at day 14. Arginase II mRNA, which was present at a low level in
untreated mice, began to increase on the third day and reached a maximum level
at day 10. Arginase I and II mRNAs were induced somewhat earlier than
collagen I mRNA. ODC mRNA, which was present at a low level in untreated mice,
began to increase on day 5, and reached a maximum level on day
10. OAT mRNA evident in untreated lungs remained little changed by
bleomycin. ODC mRNA was also induced by bleomycin treatment, whereas OAT mRNA
was highly expressed before treatment and did not change much after treatment.
These results suggest that arginase I and II are induced in the process of
lung fibrosis and supply ornithine. Then, ornithine produced by arginase is
assumed to be used for proline synthesis by OAT and for polyamine synthesis by
ODC. CAT-2 mRNA, which was weakly detectable in untreated mice, began to
increase on day 3 and increased gradually for up to 15 days.
Therefore, arginine may be derived from outside the cells by CAT-2. In
contrast, mRNA for AS, an arginine biosynthetic enzyme, was present before
treatment and remained little changed after treatment.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4. Effect of bleomycin treatment on mRNAs for collagen type I (Col I),
arginase I (AI), arginase II (AII), ODC, OAT, CAT-2, and AS in the mouse lung.
A: bleomycin was given to the mice intratracheally. After the
indicated days, total RNAs were isolated from lungs. RNAs (3.3 µg) were
subjected to blot analysis for Col I, AI, AII, ODC, OAT, CAT-2, and AS. Kidney
and liver total RNAs (2.2 µg) served as controls. The bottom panel
shows ethidium bromide staining of 28S rRNAs. B: the results of
A were quantified and are shown as means ± SD (n =
3). Maximal values are set at 100% for AI and CAT-2 mRNAs, and control values
before treatment are set at 100% for Col I, AII, ODC, OAT, and AS mRNAs.
|
|
Figure 5 shows the time
course of induction of arginase I and II proteins in the bleomycin-treated
mouse lung. Arginase I protein was not detected in untreated controls, began
to increase on day 7, increased markedly by day 10, and was
much decreased by day 14. This time of induction agrees well with
that for arginase I mRNA. The two polypeptides of arginase I apparently arose
by alternative translation initiation from the two methionine residues located
30 base pairs apart (23).
Arginase II protein was hardly detectable when lung extracts were subjected to
immunoblot analysis. Because arginase II is located in the mitochondria, we
used the mitochondrial fractions for analysis. Arginase II protein was hardly
detectable in untreated controls, began to increase on day 5, had
increased markedly by day 10, and decreased little by day
14. This time of induction agrees with that for mRNA.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5. Time course of induction of AI and AII proteins in bleomycin-treated mouse
lungs. A: bleomycin was given to mice intratracheally. After the
indicated days, lung extracts (40 µg of protein) were subjected to
immunoblot analysis with antibodies against AI (diluted 1,000-fold), Hsc70
(diluted 1,000-fold), AII (diluted 1,000-fold), and mitochondrial outer
membrane protein Tom20 (diluted 1,000-fold). Whole cell extracts were used for
AI and Hsc70, and mitochondrial extracts were used for AII and Tom20. Kidney
(mitochondrial extract) and liver (whole extract) (20 µg of protein) served
as controls. B: the results on AI and II in A were
quantified and are shown by means ± SD (n = 3). Maximal values
are set at 100%.
|
|
Induction of arginase I in macrophages and of arginase II in various
cells in bleomycin-treated mouse lungs. To identify cells expressing
arginase I and II, we performed immunohistochemical analysis of the lung
(Figs. 6 and
7). As mentioned above, the
dose of bleomycin used in these experiments is sublethal. Therefore, the
extent of induction of arginine metabolic enzymes may be underestimated.
However, we believe that our conclusion is not affected. Arginase I
immunoreactivity was undetectable in the control lung
(Fig. 6A), whereas
strong immunoreactivity was seen in the bleomycin-treated mouse lung. Types I
and II lung epithelial cells were negative for arginase I, and positive cells
were morphologically considered to be macrophages. Double immunostaining with
anti-arginase I and macrophage-specific antibodies showed that arginase I is
induced in a group of macrophages (Fig.
6B). Arginase II immunoreactivity was weakly evident in
some cells in control lungs yet was strong in many cells in bleomycin-treated
lung, apparently including type I and II epithelial cells
(Fig. 7A). Double
immunostaining with anti-arginase II and macrophage-specific antibodies showed
that arginase II is induced in macrophages but is also induced in many other
cells (Fig. 7B).
-Smooth muscle actin is a marker of myofibroblasts that plays an
important role for the development of the fibrotic lesion
(Fig. 7C).
-Smooth muscle actin staining was not detected in control mouse lung,
and arginase II immunoreactivity was evident in a small number of cells. In
bleomycin-treated lung, many cells positive for
-smooth muscle actin
staining were observed. Some of them were positive for arginase II, whereas
others were negative. These results also indicate that arginase II is induced
in many cells other than macrophages and fibroblasts.

View larger version (78K):
[in this window]
[in a new window]
|
Fig. 6. Immunostaining of the control and bleomycin-treated mouse lungs with
antibodies against AI and macrophage-specific antibody F4/80. A:
lungs of control (a) and bleomycin-treated mouse (10 days)
(b) were immunostained with an antibody against AI (diluted
1,000-fold). B: lung of bleomycin-treated mouse (10 days) was
double-stained with antibodies against AI (diluted 1,000-fold) and
macrophage-specific antibody F4/80. F4/80 staining (a), AI staining
(b), their merged image (c), and phase-contrast image
(d) of the same fields are shown. Note that AI is located in the
cytosol whereas F4/80 antigen is located in the cell membrane. Original
magnification: x200.
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 7. Immunostaining of control and bleomycin-treated mouse lungs with antibodies
against AII, macrophage-specific antibody F4/80, -smooth muscle actin,
and heat shock protein (HSP) 47. A: lungs of control (a) and
bleomycin-treated mouse (10 days) (b) were immunostained with an
antibody against AII (diluted 200-fold). B: lung of bleomycin-treated
mouse (10 days) was double-stained with antibodies against AII (diluted
200-fold) and macrophage-specific antibody F4/80. F4/80 staining (a),
arginase II staining (b), and their merged image (c) of the
same fields are shown. C: lungs of control (ad) and
bleomycin-treated (eh) mouse (10 days) were double-stained
with antibodies against AII (diluted 200-fold) and -smooth muscle actin
(diluted 500-fold). -Smooth muscle actin staining (b, f), AII
staining (c, g), their merged images (d, h), and
phase-contrast images (a, e) of the same fields are shown. Original
magnification: x200. D: lung of control (a, b) and
bleomycin-treated mouse (10 days) (c, d) were immunostained with
antibodies against AII (diluted 200-fold) (a, c) and HSP47 (b,
d), a collagen-binding chaperone protein.
|
|
HSP47 is a collagen-specific molecular chaperone in the endoplasmic
reticulum that is involved in synthesis, folding, and assembly of various
collagens, and its expression parallels that of collagen
(36). Therefore, we performed
double immunostaining of arginase II and HSP47, to see whether arginase
II-expressing cells produce collagen (Fig.
7D).
Immunostaining with anti-HSP47 revealed that the HSP47 is expressed in
various cells in lungs and that it is clearly induced with
bleomycin-treatment. Distribution of bleomycin-induced arginase II was similar
with that for HSP47. Our observations suggest that arginase II is induced in
collagen producing cells and that this enzyme provides the ornithine needed
for proline synthesis.
 |
DISCUSSION
|
---|
Arginine is a precursor for the synthesis of proline and polyamines, and
because proline is an important component of collagen, the supply of proline
can be a crucial factor in the process of lung fibrosis. Other investigators
have reported the involvement of arginine metabolic enzymes in the production
of proline or polyamines (11,
19). The successful healing of
wounds requires the local synthesis of significant amounts of collagen. Albina
et al. (2) showed that
ornithine may contribute to the synthesis of protein-incorporated proline in
wounds by increasing the extracellular pool of free proline. Arginase I and II
knockout mice were recently generated. In arginase I knockout mice, the plasma
arginine level is fourfold greater than that for wild type, and the plasma
proline level is approximately one-third for wild type
(21). In arginase II knockout
mice, the plasma arginine level is twofold greater than that for wild type,
and the plasma proline level is not changed
(42). These results show that
arginase regulates plasma arginine and proline levels. Wei et al.
(53) reported that arginase I
plays a potentially important role in controlling proliferation of rat aortic
smooth muscle cells by providing ornithine for the production of polyamines.
Our present data show that mRNA for ODC, a rate-limiting enzyme in polyamine
synthesis, was induced by bleomycin treatment. Therefore, we propose that ODC
is induced to provide polyamines for the proliferation of cells such as
fibroblasts during processes of tissue repair.
Under normal conditions, arginase I is expressed almost exclusively in the
cytosol of hepatocytes, where it serves as the final enzyme of the urea cycle.
Expression of arginase I in the liver is regulated by dietary protein
(33) and by hormones such as
glucagon and glucocorticoids
(46). More recently, arginase
I was found to be induced also by LPS, interleukin (IL)-4, cAMP, and hypoxia
in macrophages (26,
27,
34,
51) and by LPS in macrophages
of various tissues of rats and mice
(40,
45). IL-4 is also known to
induce collagen synthesis in fibroblasts in vitro
(31). On the other hand, lung
IL-4 expression was detected in a murine model of bleomycin-induced pulmonary
fibrosis (12). These results
suggest a potential role for IL-4 in pulmonary fibrosis. Perhaps because of
the potential to stimulate and amplify inflammatory responses, IL-4 stimulates
collagen synthesis in lung cells and thus promotes the progression of fibrosis
in the end stage of lung disease. If this proposal is tenable, bleomycin may
induce arginase I through the induction of IL-4.
Transforming growth factor (TGF)-
is known to enhance the expression
of the lung matrix that causes fibrosis. Boutard et al.
(4) reported that TGF-
stimulates arginase activity in macrophages. Durante et al.
(11) found that TGF-
stimulates arginine transport and metabolism in vascular smooth muscle cells.
Induction of TGF-
in the bleomycin-treated lung has been repeatedly
reported. Therefore, TGF-
is a good candidate of arginase-inducing
cytokines.
We observed that arginase I is induced in alveolar macrophages by bleomycin
treatment. Although there have been reports
(18,
41) concerning the expression
of arginase in alveolar macrophages, this is apparently the first report
showing a relationship between arginine metabolic enzymes and
bleomycin-treated lung fibrosis. In the process of fibrosis, the demand for
proline increases as it is required for the synthesis of collagen fiber.
Arginase I in alveolar macrophages may contribute to the supply of proline for
collagen synthesis in collagen-synthesizing cells. It is also possible that
arginase I supplies ornithine for synthesis of polyamines, which were found to
be required for the functional activation of macrophages
(29).
Arginase II is primarily expressed in the kidney, small intestine, and
lactating mammary glands and at low levels in other tissues, under normal
conditions (38). Arginase II,
like arginase I, is induced in LPS-activated mouse peritoneal macrophages
(34,
40). In contrast to arginase
I, arginase II expression is not increased by IL-4
(26,
27). The expressions of
arginase I and II are regulated differentially. We found that arginase II is
induced in the mouse lung after bleomycin-treatment. Induced arginase II is
present in various cells including alveolar macrophages and epithelial cells.
Expression of HSP47 in macrophages has been reported
(1). Expression of HSP47 always
parallels that of collagen in developing tissues and various cell lines and in
collagen-related pathological conditions such as fibrosis
(35). The appearance of
myofibroblasts (characterized by
-smooth muscle actin expression) is
characteristic in lung fibrotic lesions. We showed that major portions of
myofibroblasts are positive for arginase II. The distribution of HSP47
(39) is similar to that of
arginase II. These results suggest strongly that induced arginase II plays an
important role in collagen synthesis by providing proline in the
bleomycin-treated mouse lung.
IPF is a rapidly progressive illness of unknown cause, and no effective
drug therapy is currently available. Our findings suggest that arginase may
prove to be a new target for the prevention of the development of lung
fibrosis.
 |
ACKNOWLEDGMENTS
|
---|
We thank Masato Yano in our laboratory for the anti-Tom20 antibody, our
colleagues for comments and discussion, and M. Ohara (Fukuoka) for comments on
the manuscript.
DISCLOSURES
This work was supported in part by Grants-in-Aid (14370047 to M. Mori and
13670124 to T. Gotoh) from the Ministry of Education, Science, Technology,
Sports, and Culture of Japan.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: T. Gotoh, Dept. of
Molecular Genetics, Kumamoto Univ. School of Medicine, Honjo 2-2-1, Kumamoto
860-0811, Japan (E-mail:
tomomi{at}gpo.kumamoto-u.ac.jp).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
 |
REFERENCES
|
---|
- Abe K, Ozono Y,
Miyazaki M, Koji T, Shioshita K, Furusu A, Tsukasaki S, Matsuya F, Hosokawa N,
Harada T, Taguchi T, Nagata K, and Kohno S. Interstitial expression of
heat shock protein 47 and alpha-smooth muscle actin in renal allo-graft
failure. Nephrol Dial Transplant
15: 529535,
2000.[Abstract/Free Full Text]
- Albina JE,
Abate JA, and Mastrofrancesco B. Role of ornithine as a proline precursor
in healing wounds. J Surg Res
55: 97102,
1993.[ISI][Medline]
- Aso Y, Yoneda
K, and Kikkawa Y. Morphologic and biochemical study of pulmonary changes
induced by bleomycin in mice. Lab Invest
35: 558568,
1976.[ISI][Medline]
- Boutard V,
Havouis R, Fouqueray B, Philippe C, Moulinoux JP, and Baud L. Transforming
growth factor-beta stimulates arginase activity in macrophages. Implications
for the regulation of macrophage cytotoxicity. J
Immunol 155:
20772084, 1995.[Abstract]
- Bowden DH.
Unraveling pulmonary fibrosis: the bleomycin model. Lab
Invest 50:
487488, 1984.[ISI][Medline]
- Cates GA,
Nandan D, Brickenden AM, and Sanwal BD. Differentiation defective mutants
of skeletal myoblasts altered in a gelatin-binding glycoprotein.
Biochem Cell Biol 65:
767775, 1987.[ISI][Medline]
- Chandler DB,
Hyde DM, and Giri SN. Morphometric estimates of infiltrative cellular
changes during the development of bleomycin-induced pulmonary fibrosis in
hamsters. Am J Pathol 112:
170177, 1983.[Abstract]
- Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal Biochem
162: 156159,
1987.[ISI][Medline]
- Cooper JA Jr,
White DA, and Matthay RA. Drug-induced pulmonary disease. Part 1:
cytotoxic drugs. Am Rev Respir Dis
133: 321340,
1986.[ISI][Medline]
- Durante W, Liao
L, Reyna SV, Peyton KJ, and Schafer AI. Physiological cyclic stretch
directs L-arginine transport and metabolism to collagen synthesis
in vascular smooth muscle. FASEB J
14: 17751783,
2000.[Abstract/Free Full Text]
- Durante W, Liao
L, Reyna SV, Peyton KJ, and Schafer AI. Transforming growth factor-beta(1)
stimulates L-arginine transport and metabolism in vascular smooth
muscle cells: role in polyamine and collagen synthesis.
Circulation 103:
11211127, 2001.[Abstract/Free Full Text]
- Gharaee-Kermani M, Nozaki Y, Hatano K, and Phan SH. Lung
interleukin-4 gene expression in a murine model of bleomycin-induced pulmonary
fibrosis. Cytokine 15:
138147, 2001.[ISI][Medline]
- Gillette JH and
Mitchell JL. Ornithine decarboxylase: a biochemical marker of repair in
damaged tissue. Life Sci 48:
15011510, 1991.[ISI][Medline]
- Gotoh T,
Chowdhury S, Takiguchi M, and Mori M. The glucocorticoid-responsive gene
cascade. Activation of the rat arginase gene through induction of C/EBPbeta.
J Biol Chem 272:
36943698, 1997.[Abstract/Free Full Text]
- Gotoh T and
Mori M. Arginase II downregulates nitric oxide (NO) production and
prevents NO-mediated apoptosis in murine macrophage-derived RAW 264.7 cells.
J Cell Biol 144:
427434, 1999.[Abstract/Free Full Text]
- Gotoh T, Sonoki
T, Nagasaki A, Terada K, Takiguchi M, and Mori M. Molecular cloning of
cDNA for nonhepatic mitochondrial arginase (arginase II) and comparison of its
induction with nitric oxide synthase in a murine macrophage-like cell line.
FEBS Lett 395:
119122, 1996.[ISI][Medline]
- Gross TJ and
Hunninghake GW. Idiopathic pulmonary fibrosis. N Engl J
Med 345:
517525, 2001.[Free Full Text]
- Hammermann RHJ,
Hey C, Mossner J, Folkerts G, Nijkamp FP, Wessler I, and Racke K. Cationic
proteins inhibit L-arginine uptake in rat alveolar macrophages and
tracheal epithelial cells. Implications for nitric oxide synthesis.
Am J Respir Cell Mol Biol 21:
155162, 1999.[Abstract/Free Full Text]
- Ignarro LJ,
Buga GM, Wei LH, Bauer PM, Wu G, and del Soldato P. Role of the
arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell
proliferation. Proc Natl Acad Sci USA
98: 42024208,
2001.[Abstract/Free Full Text]
- Ikemoto M,
Tabata M, Miyake T, Kono T, Mori M, Totani M, and Murachi T. Expression of
human liver arginase in Escherichia coli. Purification and properties
of the product. Biochem J 270:
697703, 1990.[ISI][Medline]
- Iyer RK, Yoo
PK, Kern RM, Rozengurt N, Tsoa R, O'Brien WE, Yu H, Grody WW, and Cederbaum
SD. Mouse model for human arginase deficiency. Mol Cell
Biol 22:
44914498, 2002.[Abstract/Free Full Text]
- Janne J,
Alhonen L, and Leinonen P. Polyamines: from molecular biology to clinical
applications. Ann Med 23:
241259, 1991.[ISI][Medline]
- Kawamoto S,
Amaya Y, Murakami K, Tokunaga F, Iwanaga S, Kobayashi K, Saheki T, Kimura S,
and Mori M. Complete nucleotide sequence of cDNA and deduced amino acid
sequence of rat liver arginase. J Biol Chem
262: 62806283,
1987.[Abstract/Free Full Text]
- Kimura T,
Chowdhury S, Tanaka T, Shimizu A, Iwase K, Oyadomari S, Gotoh T, Matsuzaki H,
Mori M, Akira S, and Takiguchi M. CCAAT/enhancer-binding protein beta is
required for activation of genes for ornithine cycle enzymes by
glucocorticoids and glucagon in primary-cultured hepatocytes. FEBS
Lett 494:
105111, 2001.[ISI][Medline]
- Kimura T,
Christoffels VM, Chowdhury S, Iwase K, Matsuzaki H, Mori M, Lamers WH,
Darlington GJ, and Takiguchi M. Hypoglycemia-associated hyperammonemia
caused by impaired expression of ornithine cycle enzyme genesin C/EBPalpha
knockout mice. J Biol Chem 273:
2750527510, 1998.[Abstract/Free Full Text]
- Louis CA, Mody
V, Henry WL Jr, Reichner JS, and Albina JE. Regulation of arginase
isoforms I and II by IL-4 in cultured murine peritoneal macrophages.
Am J Physiol Regul Integr Comp Physiol
276: R237R242,
1999.[Abstract/Free Full Text]
- Louis CA,
Reichner JS, Henry WL Jr, Mastrofrancesco B, Gotoh T, Mori M, and Albina
JE. Distinct arginase isoforms expressed in primary and transformed
macrophages: regulation by oxygen tension. Am J Physiol Regul
Integr Comp Physiol 274:
R775R782, 1998.[Abstract/Free Full Text]
- McKnight AJ,
Macfarlane AJ, Dri P, Turley L, Willis AC, and Gordon S. Molecular cloning
of F4/80, a murine macrophage-restricted cell surface glycoprotein with
homology to the G-protein-linked transmembrane 7 hormone receptor family.
J Biol Chem 271:
486489, 1996.[Abstract/Free Full Text]
- Messina L,
Spampinato G, Arcidiacono A, Malaguarnera L, Pagano M, Kaminska B, Kaczmarek
L, and Messina A. Polyamine involvement in functional activation of human
macrophages. J Leukoc Biol 52:
585587, 1992.[Abstract]
- Modolell M,
Corraliza IM, Link F, Soler G, and Eichmann K. Reciprocal regulation of
the nitric oxide synthase/arginase balance in mouse bone marrow-derived
macrophages by TH1 and TH2 cytokines. Eur J Immunol
25: 11011104,
1995.[ISI][Medline]
- Monroe JG,
Haldar S, Prystowsky MB, and Lammie P. Lymphokine regulation of
inflammatory processes: interleukin-4 stimulates fibroblast proliferation.
Clin Immunol Immunopathol 49:
292298, 1988.[ISI][Medline]
- Mori M and
Gotoh T. Regulation of nitric oxide production by arginine metabolic
enzymes. Biochem Biophys Res Commun
275: 715719,
2000.[ISI][Medline]
- Morris SM Jr. Regulation of enzymes of urea and arginine synthesis. Annu
Rev Nutr 12:
81101, 1992.[ISI][Medline]
- Morris SM Jr,
Kepka-Lenhart D, and Chen LC. Differential regulation of arginases and
inducible nitric oxide synthase in murine macrophage cells. Am J
Physiol Endocrinol Metab 275:
E740E747, 1998.[Abstract/Free Full Text]
- Nagata K.
Hsp47: a collagen-specific molecular chaperone. Trends Biochem
Sci 21:
2226, 1996.[Medline]
- Nakai A, Satoh
M, Hirayoshi K, and Nagata K. Involvement of the stress protein HSP47 in
procollagen processing in the endoplasmic reticulum. J Cell
Biol 117:
903914, 1992.[Abstract]
- Osanai K,
Takahashi K, Sato S, Iwabuchi K, Ohtake K, Sata M, and Yasui S. Changes of
lung surfactant and pressure-volume curve in bleomycin-induced pulmonary
fibrosis. J Appl Physiol 70:
13001308, 1991.[Abstract/Free Full Text]
- Ozaki M, Gotoh
T, Nagasaki A, Miyanaka K, Takeya M, Fujiyama S, Tomita K, and Mori M.
Expression of arginase II and related enzymes in the rat small intestine and
kidney. J Biochem (Tokyo) 125:
586593, 1999.[Abstract]
- Razzaque MS and
Taguchi T. Role of glomerular epithelial cell-derived heat shock protein
47 in experimental lipid nephropathy. Kidney Int Suppl
71: S256S259,
1999.[Medline]
- Salimuddin, Nagasaki A,
Gotoh T, Isobe H, and Mori M. Regulation of the genes for arginase
isoforms and related enzymes in mouse macrophages by lipopolysaccharide.
Am J Physiol Endocrinol Metab
277: E110E117,
1999.[Abstract/Free Full Text]
- Shearer JD,
Richards JR, Mills CD, and Caldwell MD. Differential regulation of
macrophage arginine metabolism: a proposed role in wound healing.
Am J Physiol Endocrinol Metab
272: E181E190,
1997.[Abstract/Free Full Text]
- Shi O, Morris
SM Jr, Zoghbi H, Porter CW and O'Brien WE. Generation of a mouse model for
arginase II deficiency by targeted disruption of the arginase II gene.
Mol Cell Biol 21:
811813, 2001.[Abstract/Free Full Text]
- Snider GL,
Celli BR, Goldstein RH, O'Brien JJ, and Lucey EC. Chronic interstitial
pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Lung
volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood
gas studied. Am Rev Respir Dis
117: 289297,
1978.[ISI][Medline]
- Snider GL,
Hayes JA, and Korthy AL. Chronic interstitial pulmonary fibrosis produced
in hamsters by endotracheal bleomycin: pathology and stereology. Am
Rev Respir Dis 117:
10991108, 1978.[ISI][Medline]
- Sonoki T,
Nagasaki A, Gotoh T, Takiguchi M, Takeya M, Matsuzaki H, and Mori M.
Coinduction of nitric-oxide synthase and arginase I in cultured rat peritoneal
macrophages and rat tissues in vivo by lipopolysaccharide. J Biol
Chem 272:
36893693, 1997.[Abstract/Free Full Text]
- Takiguchi M and
Mori M. Transcriptional regulation of genes for ornithine cycle enzymes.
Biochem J 312:
649659, 1995.[ISI][Medline]
- Thrall RS,
McCormick JR, Jack RM, McReynolds RA, and Ward PA. Bleomycin-induced
pulmonary fibrosis in the rat: inhibition by indomethacin. Am J
Pathol 95:
117130, 1979.[Abstract]
- Thrall RS,
Swendsen CL, Shannon TH, Kennedy CA, Frederick DS, Grunze MF, and Sulavik
SB. Correlation of changes in pulmonary surfactant phospholipids with
compliance in bleomycin-induced pulmonary fibrosis in the rat. Am
Rev Respir Dis 136:
113118, 1987.[ISI][Medline]
- Usuki J and
Fukuda Y. Evolution of three patterns of intra-alveolar fibrosis produced
by bleomycin in rats. Pathol Int
45: 552564,
1995.[ISI][Medline]
- Waddington SN,
Tam FW, Cook HT, and Cattell V. Arginase activity is modulated by IL-4 and
HOArg in nephritic glomeruli and mesangial cells. Am J Physiol
Renal Physiol 274:
F473F480, 1998.[Abstract/Free Full Text]
- Wang WW,
Jenkinson CP, Griscavage JM, Kern RM, Arabolos NS, Byrns RE, Cederbaum SD, and
Ignarro LJ. Co-induction of arginase and nitric oxide synthase in murine
macrophages activated by lipopolysaccharide. Biochem Biophys Res
Commun 210:
10091016, 1995.[ISI][Medline]
- Watanabe Y and
Komatsu K. Biomechanical and morphological studies on the periodontal
ligament of the rat molar after treatment with alpha-amylase in vitro.
Connect Tissue Res 36:
3549, 1997.[ISI][Medline]
- Wei LH, Wu G,
Morris SM Jr, and Ignarro LJ. Elevated arginase I expression in rat aortic
smooth muscle cells increases cell proliferation. Proc Natl Acad
Sci USA 98:
92609264, 2001.[Abstract/Free Full Text]
- Woessner JF. The determination of hydroxyproline in tissue
and protein samples containing small proportions of this amino acid.
Arch Biochem Biophys 93:
440447, 1961.[ISI]
- Yano M,
Kanazawa M, Terada K, Takeya M, Hoogenraad N, and Mori M. Functional
analysis of human mitochondrial receptor Tom20 for protein import into
mitochondria. J Biol Chem 273:
2684426851, 1998.[Abstract/Free Full Text]
- Yu Y, Terada K,
Nagasaki A, Takiguchi M, and Mori M. Preparation of recombinant
argininosuccinate synthetase and argininosuccinate lyase: expression of the
enzymes in rat tissues. J Biochem (Tokyo)
117: 952957,
1995.[Abstract]
- Zia S, Hyde DM,
and Giri SN. Development of a bleomycin hamster model of subchronic lung
fibrosis. Pathology 24:
155163, 1992.[ISI][Medline]