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J Biol Chem, Vol. 275, Issue 17, 12475-12480, April 28, 2000
Des-Arg10-kallidin Engagement of the B1 Receptor
Stimulates Type I Collagen Synthesis via Stabilization of
Connective Tissue Growth Factor mRNA*
Dennis A.
Ricupero §,
Jose R.
Romero¶ ,
David C.
Rishikof **, and
Ronald H.
Goldstein
From the Pulmonary Center, Departments of Medicine
and Biochemistry, Boston University School of Medicine and the
Boston Veterans Affairs Medical Center and the
¶ Endocrine-Hypertension Division, Department of Medicine, Harvard
Medical School, Boston, Massachusetts 02118-2394
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ABSTRACT |
Expression of the kinin B1 receptor is
up-regulated in chronic inflammatory and fibrotic disorders; however,
little is known about its role in fibrogenesis. We examined human
embryonic lung fibroblasts that constitutively express the B1 receptor
and report that engagement of the B1 receptor by
des-Arg10-kallidin stabilized connective tissue
growth factor (CTGF) mRNA, stimulated an increase in 1(I)
collagen mRNA, and stimulated type I collagen production. These
events were not observed in B2 receptor-activated fibroblasts. In
addition, B1 receptor activation by des-Arg10-kallidin
induced a rise in cytosolic Ca2+ that is consistent with B1
receptor pharmacology. Our results show that the
des-Arg10-kallidin-stimulated increase in 1(I) collagen
mRNA was time- and dose-dependent, with a peak response
observed at 20 h with 100 nM
des-Arg10-kallidin. The increase in CTGF mRNA was also
time- and dose-dependent, with a peak response observed at
4 h with 100 nM des-Arg10-kallidin. The
increase in CTGF mRNA was blocked by the B1 receptor antagonist
des-Arg10,Leu9-kallidin. Inhibition of protein
synthesis by cycloheximide did not block the
des-Arg10-kallidin-induced increase in CTGF mRNA. These
results suggest that engagement of the kinin B1 receptor contributes to
fibrogenesis through increased expression of CTGF.
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INTRODUCTION |
Kinins are involved in the regulation of a variety of
physiological and cellular functions, including smooth muscle tone, pain perception, inflammation, and cellular proliferation (1, 2).
Molecular biological and pharmacological studies have identified two
G-protein-coupled kinin receptors, B1 and B2 (3-5). Activation of the
B1 receptor induces cellular and physiological responses that often
mimic the responses observed following activation of the B2 receptor.
For instance, both receptors activate nuclear factor B in
fibroblasts (6), modulate vascular tone (7), and activate phospholipase
C in mesangial cells (8). However, receptor-specific physiological
responses induced by the kinin receptors are demonstrated in the
kinin-mediated bronchoconstriction of asthmatic airways. The B2
receptor agonists bradykinin
(BK)1 and kallidin (Lys-BK)
are potent bronchoconstrictors (9), whereas the B1 receptor agonist
des-Arg10-kallidin does not induce bronchoconstriction
(10).
The B2 receptor, the classical bradykinin receptor, is widely expressed
and binds both BK and kallidin with high affinity. In wound repair,
activation of the B2 receptor induces a variety of effects, including
increased neutrophil proliferation, stimulation of macrophage
spreading, release of histamine from mast cells, synthesis of
platelet-activating factor and prostaglandins in endothelial cells,
release of tachykinin and acetylcholine from sensory nerve endings,
increased microvascular permeability, and fibroblast proliferation
(2).
Early studies described the B1 receptor as an inducible receptor whose
expression is up-regulated following exposure to interleukin-1 or
other pro-inflammatory agents (11). A recent study has identified expression of the B1 receptor by immunohistochemistry in transbronchial biopsies from patients with sarcoidosis or progressive systemic sclerosis, whereas expression of the B1 receptor was undetectable in
normal subjects (12). Activation of the B1 receptor induces relaxation
or contraction of various smooth muscle cell preparations, mediates
chronic pain, and may be involved in chronic inflammation (1). Recent
studies have demonstrated the expression of the B1 receptor in
apparently healthy renal (13), intestinal (14), ocular (15), stomach
(16), and pulmonary (17) tissues; however, the role of the B1 receptor
in homeostasis and wound repair is not understood. Activation of the B1
receptor stimulates prostaglandin synthesis in fibroblasts, release of
tumor necrosis factor and interleukin-1 from macrophages, and
prostaglandin and platelet-activating factor synthesis in endothelial
cells (1).
Recently, it was proposed that TGF- stimulates fibrogenesis and
proliferation in fibroblasts via a mechanism that requires de
novo synthesis of a secondary factor, which has been identified as
connective tissue growth factor (CTGF) (18). CTGF, a member of the CCN
family, is a cysteine-rich, heparin-binding, 349-amino acid protein
(19). Other members of the CCN family include Fisp12/BIGM2 (mouse
ortholog of CTGF) (20), Cyr61 (and the chick ortholog Cef10) (21), Nov
(human and Xenopus orthologs) (22), Elm-1 (23), CTGF-L (24),
and WISP-3 (25). CCN proteins possess a secretory signal peptide and
four distinct protein modules: an insulin-like growth factor-binding
domain, a von Willebrand factor type C repeat, a thrombospondin type 1 repeat, and a C-terminal module. CTGF-L does not contain the C-terminal
domain. The CCN family is further distinguished by the high degree of
amino acid homology (50-90%) and conservation of 38 cysteine
residues. TGF- (but not epidermal growth factor, fibroblast growth
factor, or platelet-derived growth factor) stimulates CTGF
transcription in normal rat kidney fibroblasts (26). In adult mammals,
CTGF is expressed in high levels during wound repair and at sites of connective tissue formation in a variety of fibrotic disorders (27,
28). CTGF is expressed in lung fibroblasts and has been suggested to
play a role in the pathogenesis of lung fibrosis (29).
Our goal was to examine B1 receptor-induced fibrogenesis. Using a human
embryonic lung fibroblast cell line, we report that des-Arg10-kallidin, upon binding to the B1 receptor,
activates an increase in cytosolic Ca2+ with a different
fluorescence signature using fura-2 than the B2 receptor and
specifically induces dose- and time-dependent increases in
1(I) collagen mRNA and CTGF mRNA. The increase in steady-state CTGF mRNA levels involves stabilization of the message through a mechanism that is cycloheximide-insensitive and
12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive. The
data suggest that the B1 receptor has a distinct function in the
pathophysiology of fibrotic lung diseases.
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MATERIALS AND METHODS |
Tissue Culture--
Human embryonic lung fibroblasts (IMR-90,
Institute for Medical Research, Camden, NJ) were grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 0.37 g/100 ml sodium
bicarbonate, 10% (v/v) fetal bovine serum (FBS), 100 units/ml
penicillin, 10 µg/ml streptomycin, 0.1 mM sodium
pyruvate, and 0.1 mM nonessential amino acids. Cell numbers
were determined by triplicate cell counts with an electronic particle
counter (Coulter Counter ZM).
Northern Blotting--
Confluent IMR-90 fibroblast cultures were
incubated in DMEM supplemented with 0.4% FBS for 24 h. The
culture medium was supplemented with protease inhibitors (captopril (10 µM), phosphoramidon (1 µM), and
DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (1 µM)) and stimulated as indicated. Total cellular RNA was
isolated by the single-step method employing guanidine
thiocyanate/phenol/chloroform extraction as described by Chomczynski
and Sacchi (30). RNA was quantified by absorbance at 260 nm. Purity was
determined by absorbance at 280 and 310 nm. RNA (10 µg) was
electrophoresed through a 6% formaldehyde-containing 1% agarose gel
and transferred to a nylon membrane. RNA loading was assessed by
ethidium bromide staining of ribosomal bands and by co-hybridization
with glyceraldehyde-3-phosphate dehydrogenase. The 1(I) collagen
probe is a 1.5-kilobase pair fragment of rat 1(I) collagen cDNA
that specifically binds human 1(I) collagen mRNA. The CTGF probe
is a 586-base pair polymerase chain reaction product generated with the
forward primer gtggagtatgtaccgacggcc and the reverse primer
acaggcaggtcagtgagcacgc. The filter was exposed to x-ray film for
autoradiography at several different times to ensure that the bands
could be quantified by densitometry within the linear range.
Bioassay for TGF- Activity--
Quantification of TGF-
released by IMR-90 fibroblast cultures was determined using a TGF-
bioassay based on the inhibition of growth of mink lung epithelial
cells (MLECs) by TGF- (31). MLECs were maintained in DMEM
supplemented with 10% FBS and passaged every 3 days. MLECs were seeded
at a density of 2 × 105 cells/well in 24-well plates.
After 24 h, the cultures were washed twice with phosphate-buffered
saline and refed with DMEM (0.5 ml/well) without FBS and supplemented
with leupeptin (2 µg/ml), aprotinin (2 µg/ml), and pepstatin A (2 µg/ml). Conditioned medium from control or
des-Arg10-kallidin-stimulated IMR-90 fibroblasts was
centrifuged (500 × g, 10 min) to remove non-adherent
cells and then added to the MLECs (0.5 ml/well). The cultures were
incubated for 20 h at 37 °C and 5% CO2 and then
pulsed with [3H]thymidine (1 µCi/well; NEN Life Science
Products) for 2 h. The plates were washed twice with cold
phosphate-buffered saline, fixed with 1 ml of acetic acid/methanol
(1:3, v/v) 1 h at room temperature, washed twice with 80%
methanol, digested for 30 min with 0.5 ml of 0.05% trypsin dissolved
with 0.5 ml of 0.1% SDS, and scintillation-counted. The quantification
of the TGF- content of conditioned medium was interpolated from a
standard curve generated by replacing conditioned medium with DMEM
supplemented with TGF- (0.003-1.0 ng/ml). Inhibition of MLEC
incorporation of [3H]thymidine ranged from 92% (0.25 ng/ml) to 0.3% (0.003 ng/ml), with an IC50 of ~0.15
ng/ml.
Measurements of Cytosolic Calcium--
Steady-state and
agonist-stimulated levels of cytosolic Ca2+ in IMR-90
fibroblasts were determined with the fluorescent probe fura-2. Briefly,
IMR-90 cells were grown to confluence on glass coverslips. The cells
were then loaded with fura-2/AM (5 µM) for 30 min at
37 °C in physiological saline solution containing 140 mmol/liter
NaCl, 5 mmol/liter KCl, 10 mmol/liter HEPES (pH 7.4), 1 mmol/liter
NaHPO4, 1 mmol/liter CaCl2, 1 MgSO4, and 5 mmol/liter glucose. Cells were washed twice
with physiological saline solution without fura-2. The cell-containing
coverslips were then placed diagonally in a quartz cuvette that
contained physiological saline solution (37 °C), and cytosolic
Ca2+ was monitored in real time for ~15 min as described
by Fajtova et al. (32) with modifications (33). Fura-2
fluorescence was recorded at 510 nm emission wavelength with excitation
monochrometers centered at 350 and 380 nm (in a PTI
spectrofluorometer). Stable values were obtained for periods of up to
20 min. Cell fluorescence was corrected for autofluorescence, and the
contribution of dye leakage was estimated at <2% with
Mn2+.
Assay for Collagen Synthesis--
Confluent quiescent fibroblast
cultures were stimulated with 100 nM
des-Arg10-kallidin, 100 nM bradykinin, or 1 ng/ml TGF- as indicated for 16 h in serum-free DMEM
supplemented with [3H]proline (2 µCi/ml) and ascorbate
(50 µg/ml). The cultures were scraped into phosphate-buffered saline
with EDTA (2 mM), phenylmethylsulfonyl fluoride (100 µM), and hydoxymercuribenzoate (10 µM). The
lysates were dialyzed at 4 °C for 48 h, lyophilized, digested
with pepsin (10 µg/ml) in 0.5 N acetic acid for 16 h
at 4 °C, dialyzed for 48 h at 4 °C, and lyophilized (34).
The digests were separated on a 6% polyacrylamide gel. Autoradiography
was performed as described (35).
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RESULTS |
Embryonic human lung fibroblasts (IMR-90) constitutively express
both the B1 and B2 receptors (36). Since activation of the B1 or B2
receptor activates phospholipase C and induces a rise in cytosolic
Ca2+ in a number of cell types (37, 38), we monitored
cytosolic Ca2+ levels in kinin-stimulated fibroblasts. To
activate the B2 receptor, we employed BK or kallidin, both of which are
nearly indistinguishable pharmacologically and physiologically. To
activate the B1 receptor, we used des-Arg10-kallidin (39).
We found that activation of the B1 receptor by
des-Arg10-kallidin induced a dose-dependent
increase in cytosolic Ca2+ in human lung fibroblasts (Fig.
1). Increases in cytosolic
Ca2+ were detected at the threshold concentration of 1 nM des-Arg10-kallidin. The maximal increase in
cytosolic Ca2+ induced by des-Arg10-kallidin
was observed at 100 nM (EC50 = 1.9 nM; r2 = 0.9871). Activation of the
B2 receptor induced an increase in cytosolic Ca2+ that was
also dose-dependent (EC50 = 0.7 nM;
r2 = 0.9763) (data not shown). To examine the
pharmacology of the kinin receptors, we monitored cytosolic
Ca2+ concentrations in kinin-stimulated fibroblasts. In
fibroblasts stimulated with the B1 receptor agonist
des-Arg10-kallidin, the cytosolic Ca2+
increased gradually and returned to base-line levels (Fig.
2A). The B1 receptor
antagonist des-Arg10,Leu9-kallidin did not
induce an increase in cytosolic Ca2+, but did block the
increase in cytosolic Ca2+ stimulated by
des-Arg10-kallidin. However, the kallidin-stimulated
increase in cytosolic Ca2+ was not affected by
des-Arg10,Leu9-kallidin (Fig. 2B).
In fibroblasts stimulated with 1 nM kallidin, cytosolic
Ca2+ levels increased sharply and remained elevated (Fig.
2C). The B2 receptor-specific antagonist (HOE 140) did not
stimulate an increase in cytosolic Ca2+ or interfere with
the Ca2+ movement induced by
des-Arg10-kallidin. Conversely, the kallidin-stimulated
increase in cytosolic Ca2+ was blocked by 1 µM HOE 140 (Fig. 2D). We conclude that
des-Arg10-kallidin stimulates an increase in cytosolic
Ca2+ via exclusive engagement of the B1 receptor.

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Fig. 1.
Ca2+ transients in
des-Arg10-kallidin-stimulated fibroblasts. IMR-90
fibroblasts were grown on glass coverslips, incubated in DMEM
supplemented with 0.4% FBS for 24 h, and loaded with fura-2 as
described under "Materials and Methods." The fibroblasts were
stimulated with the concentrations of des-Arg10-kallidin as
indicated. The maximal 340/380 nm emission ratio increases are
presented. Results are representative of three independent
experiments.
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Fig. 2.
Effect of B1 receptor antagonist. IMR-90
fibroblasts were grown on glass coverslips, incubated in DMEM
supplemented with 0.4% FBS for 24 h, and loaded with fura-2 as
described under "Materials and Methods." The 340/380 nm emission
ratios are presented. A, the fibroblasts were stimulated
with 10 nM des-Arg10-kallidin
(arrow). Results are representative of three independent
experiments. B, the fibroblasts were stimulated with 1 µM des-Arg10,Leu9-kallidin ( ),
10 nM des-Arg10-kallidin (arrow),
and 1 nM kallidin ( ). Results are representative of
three independent experiments. C, the fibroblasts were
stimulated with 1 nM kallidin ( ). Results are
representative of three independent experiments. D, the
fibroblasts were stimulated with 1 µM HOE 140 ( ), 1 nM kallidin ( ), and 10 nM
des-Arg10-kallidin (arrow). Results are
representative of three independent experiments.
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To study the effect of kinin receptor activation on the production of
type I collagen, fibroblasts were stimulated with the B1 receptor
agonist des-Arg10-kallidin, the B2 receptor agonist BK, or
TGF- (Fig. 3). BK did not stimulate
type I collagen production, whereas des-Arg10-kallidin
stimulated an increase in type I collagen production. As previously
reported, TGF- is a potent stimulus of type I collagen production
(36). Type I collagen is composed of two 1(I) collagen peptides and
one 2(I) collagen peptide. Presented in Fig.
3 are radiolabeled 1(I) and 2(I)
collagen peptides resolved by polyacrylamide gel electrophoresis. We
determined that des-Arg10-kallidin stimulated an increase
in both 1(I) and 2(I) collagen peptides proportionally.

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Fig. 3.
Type I collagen production. IMR-90
fibroblasts were grown to confluence in 150-mm dishes; incubated in
DMEM supplemented with 0.4% FBS for 24 h; and stimulated with 100 nM BK, 100 nM
des-Arg10-kallidin (DAK), or 1 ng/ml
TGF- or unstimulated (Control) for 16 h. Type I
collagen was prepared as described under "Materials and
Methods." Results are representative of two independent
experiments.
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Fig. 4.
1(I) collagen mRNA in
kinin-stimulated fibroblasts. Confluent IMR-90 fibroblasts were
incubated in DMEM supplemented with 0.4% FBS for 24 h. Total RNA
was harvested and Northern-blotted (10 µg/lane) with probes for
1(I) collagen mRNA and GAPDH mRNA as described under
"Results." A, fibroblasts were stimulated with 100 nM des-Arg10-kallidin (DAK) or 100 nM BK or were unstimulated (Control) for 20 h. The location of the ribosomal RNA (28 S) is indicated. Results are
representative of three independent experiments. B,
fibroblasts were stimulated with concentrations of
des-Arg10-kallidin ranging from 10 12 to
10 6 M for 20 h as indicated. Results are
representative of three independent experiments. C,
fibroblasts were stimulated with 100 nM
des-Arg10-kallidin. Total RNA was isolated at the indicated
times. Results are representative of three independent
experiments.
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We examined the modulation of steady-state 1(I) collagen mRNA
levels by kinins. BK did not stimulate an increase in 1(I) collagen
mRNA. However, in fibroblasts stimulated with
des-Arg10-kallidin, there was an increase in 1(I)
collagen mRNA (Fig. 4A). Variations in RNA loading were
monitored by expression of GAPDH mRNA.
A narrow dose-response relationship was found for the
des-Arg10-kallidin-stimulated increase in 1(I) collagen
mRNA (Fig. 4B). At concentrations <1 nM
des-Arg10-kallidin, we did not detect any changes in
1(I) collagen mRNA. Maximal stimulation occurred at
concentrations >10 nM. Kinetic studies detected increases
in 1(I) collagen mRNA beginning at 4 h, with a maximum
response at 24 h (Fig. 4C). In contrast, neither des-Arg10-kallidin nor kallidin induced an increase in
fibronectin mRNA (data not shown).
TGF- mediates increases in collagen synthesis induced by other
G-protein-coupled receptor agonists, including angiotensin II (40) and
thromboxane (41). We determined the amount of active TGF- present in
the culture medium from untreated and des-Arg10-kallidin-treated fibroblasts using the bioassay
in which MLEC growth was inhibited in a dose-dependent
manner by active TGF- . The medium from
des-Arg10-kallidin-stimulated fibroblasts was added to MLEC
cultures, and [3H]thymidine incorporation was monitored
(Table I). The medium from untreated and
des-Arg10-kallidin-stimulated fibroblasts contained small
but similar amounts of TGF- . The values were interpolated from a
TGF- inhibition standard curve. The amount of active TGF- in the
medium from des-Arg10-kallidin-stimulated fibroblasts
(0.38 ± 0.053 ng/ml) was not statistically different from the
control values (p > 0.05).
Des-Arg10-kallidin (1 µM) did not alter MLEC
growth, nor did the presence of des-Arg10-kallidin (1 µM) alter TGF- -mediated growth inhibition (data not
shown).
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Table I
TGF- content of conditioned media
Confluent quiescent IMR-90 fibroblasts were washed with
phosphate-buffered saline and incubated in serum-free DMEM with or
without 100 nM des-Arg10-kallidin for 24 h.
The medium was removed, centrifuged to remove non-adherent cells, and
stored at 70 °C. The TGF- content of the conditioned medium was
determined in triplicate as described under "Results" and
reported as the mean ± S.D. for 10 independent experiments.
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To determine whether des-Arg10-kallidin amplifies the
TGF- signaling mechanism, we employed the luciferase reporter
p3TP-LUX, which is activated by TGF- via Smad signaling (42). In
fibroblasts that were transiently transfected with p3TP-LUX, TGF-
stimulated transcription from p3TP-LUX, as indicated by increased
luciferase activity. Des-Arg10-kallidin did not stimulate
an increase in luciferase activity. In cultures stimulated with both
TGF- and des-Arg10-kallidin, the luciferase activity was
comparable to the luciferase activity in cultures stimulated with
TGF- alone (Fig. 5). We conclude that
the des-Arg10-kallidin-stimulated increase in 1(I)
collagen mRNA is mediated through a mechanism that does not
increase the amount of active TGF- or amplify the intracellular
signal generated by TGF- . At this time, we do not exclude the
possibility that des-Arg10-kallidin amplifies
non-SMAD-mediated TGF- signaling.

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Fig. 5.
Luciferase activity. IMR-90 fibroblasts
were transfected with p3TP-LUX as described under "Results." The
cultures were stimulated with 100 nM
des-Arg10-kallidin (DAK), 1 ng/ml TGF-
(T), or 1 ng/ml TGF- plus 100 nM
des-Arg10-kallidin (T+DAK) for 8 h as
indicated. The luciferase activity was assayed as described under
"Results" and is reported as mean ± S.D. of triplicates.
Results are representative of three independent experiments.
C, control.
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In human lung fibroblasts, des-Arg10-kallidin stimulates
proliferation (36) and increased levels of 1(I) collagen mRNA
(Fig. 4A). Since TGF- stimulates extracellular matrix
protein synthesis and proliferation in fibroblasts through de
novo synthesis of CTGF (26), we examined the effects of kinins on
the steady-state levels of CTGF mRNA (Fig.
6A). TGF- stimulated an
increase in CTGF mRNA, and activation of the B2 receptor did not
induce a change in CTGF mRNA levels. In contrast, activation of the
B1 receptor by des-Arg10-kallidin stimulated an increase in
CTGF mRNA. As with 1(I) collagen mRNA, the
des-Arg10-kallidin-stimulated increase in CTGF mRNA was
dose-dependent (Fig. 6B). For the dose-response
studies, fibroblasts were stimulated with
des-Arg10-kallidin ranging from 10 pM to 1 µM. Maximal increases in CTGF mRNA were observed at
concentrations of des-Arg10-kallidin >10 nM.
The des-Arg10-kallidin response was further characterized
in a time course study. Total RNA was harvested at various time points
(2-24 h). Increases in CTGF mRNA levels were detected at 2 h,
with a maximum response at 4 h (Fig. 6C). At 24 h,
the level of CTGF mRNA returned to basal levels.

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Fig. 6.
CTGF mRNA levels in kinin-stimulated
fibroblasts. Confluent IMR-90 fibroblasts were incubated in DMEM
supplemented with 0.4% FBS for 24 h. Following stimulation, total
RNA was harvested and Northern-blotted (10 µg/lane) with probes for
CTGF and GAPDH as described under "Results." A, the
cultures were stimulated with 100 nM
des-Arg10-kallidin (DAK), 100 nM
kallidin (K), or 1 ng/ml TGF- or were unstimulated
(Control) for 4 h as indicated. Location of the
ribosomal RNA (18 S) is indicated. Results are representative of three
independent experiments. B, des-Arg10-kallidin
stimulated a dose-dependent increase in CTGF mRNA. The
cultures were stimulated with des-Arg10-kallidin (ranging
from 10 11 to 10 6 M) for 4 h. Results are representative of three independent experiments.
C, confluent IMR-90 fibroblasts were stimulated with 100 nM des-Arg10-kallidin for the indicated time
points. Results are representative of three independent
experiments.
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As demonstrated with the des-Arg10-kallidin-stimulated
increase in cytosolic Ca2+, the B1 receptor antagonist
des-Arg10,Leu9-kallidin attenuated the
des-Arg10-kallidin-induced increase in CTGF mRNA. In
contrast to des-Arg10-kallidin,
des-Arg10,Leu9-kallidin did not induce an
increase in CTGF mRNA (Fig.
7A). However, when the
fibroblasts were preincubated with the B1 receptor antagonist des-Arg10,Leu9-kallidin (1 µM)
and then stimulated with des-Arg10-kallidin (100 nM), the increase in CTGF mRNA was reduced.

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Fig. 7.
Modulators of
des-Arg10-kallidin-stimulated increases in CTGF
mRNA. Confluent IMR-90 fibroblasts were incubated in DMEM
supplemented with 0.4% FBS for 24 h. Following stimulation, total
RNA was harvested and Northern-blotted (10 µg/lane) with probes for
CTGF and GAPDH as described under "Results." A, the B1
receptor antagonist attenuated the
des-Arg10-kallidin-stimulated increase in CTGF mRNA.
The fibroblasts were incubated with 1 µM
des-Arg10,Leu9-kallidin (DALK) for
10 min and then stimulated with 100 nM
des-Arg10-kallidin (DAK) or 1 ng/ml TGF- for
4 h as indicated. Results are representative of three independent
experiments. B, the cultures were pretreated with
actinomycin D (5 µg/ml) for 10 min and then stimulated with 100 nM des-Arg10-kallidin or 1 ng/ml TGF- or
unstimulated for 4 h. Results are representative of three
independent experiments. C, the cultures were pretreated
with 1 µM cycloheximide for 30 min and then stimulated
with 100 nM des-Arg10-kallidin or 1 ng/ml
TGF- or were unstimulated for 6 h. Results are representative
of three independent experiments. D, the cultures were
pretreated with 100 nM TPA for 10 min and then stimulated
with 100 nM des-Arg10-kallidin or 1 ng/ml
TGF- were or unstimulated for 4 h. Results are representative
of three independent experiments.
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We previously reported that TGF- stimulates transcription of CTGF
(43). When transcription was blocked with actinomycin D, there were
reduced basal levels of CTGF mRNA, indicating that CTGF mRNA
was constitutively transcribed in quiescent fibroblasts (Fig.
7B). As previously reported, actinomycin D blocks the
TGF- -stimulated transcription of CTGF (43). However, the
des-Arg10-kallidin-stimulated increase in CTGF mRNA was
not blocked by actinomycin D, indicating that the
des-Arg10-kallidin-induced increase in CTGF mRNA is due
to stabilization of the message.
Studies on the granulocyte-macrophage colony-stimulating factor
(GM-CSF) mRNA indicated that inhibition of protein synthesis with
cycloheximide (CHX) or short-term treatment with TPA induces stabilization of GM-CSF mRNA (44). We characterized the
des-Arg10-kallidin response by blocking protein synthesis
with CHX. TGF- signaling was not affected by inhibition of protein
synthesis. In fibroblasts incubated with either CHX or
des-Arg10-kallidin, there was an increase in CTGF mRNA.
Furthermore, in fibroblasts stimulated with both CHX and
des-Arg10-kallidin, CTGF mRNA levels increased in an
additive manner, suggesting that the des-Arg10-kallidin
response does not involve inhibition of protein synthesis (Fig.
7C). We further characterized the
des-Arg10-kallidin-induced stabilization of CTGF mRNA
by pretreating the fibroblasts with TPA (100 nM, 10 min).
TPA alone did not alter the basal levels of CTGF mRNA or alter the
TGF- response. However, TPA attenuated the
des-Arg10-kallidin response (Fig. 7D).
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DISCUSSION |
In vivo, the B1 receptor is detected in low abundance
in normal tissues and is up-regulated following exposure to
pro-inflammatory agents via nuclear factor B activation. In the
studies presented here, we utilized human embryonic lung fibroblasts
that constitutively express the B1 receptor. Our results do not require
up-regulation of B1 receptor number since treatment with actinomycin D
or CHX does not inhibit the des-Arg10-kallidin-induced
increase in CTGF mRNA. Since each of these agents prevents de
novo synthesis of the B1 receptor, it appears that the number of
receptors constitutively expressed by the fibroblasts is sufficient for
signaling
Exogenous administration of B1 and B2 receptor agonists in
vivo and in vitro generates responses that appear
qualitatively similar. For example, both the B1 and B2 receptors
mediate pain perception (45); both receptors activate phospholipase C
in mesangial cells (46); and both receptors stimulate mitogenesis in
fibroblasts (36). In addition, the structural similarities of the B1
and B2 receptor agonists suggest that the kinin receptors may be
isoforms. However, our Ca2+ studies clearly demonstrate
that the physiological effects mediated by the B1 and B2 receptor
systems are distinct. These findings, together with the low homology
between the receptors (36% at the amino acid level) and the patterns
of expression, indicate that the kinin receptors have distinct
physiological and cellular responses.
Engagement of the B1 or B2 receptor affects extracellular matrix
deposition, but in different ways. Activation of the B2 receptor stimulates synthesis of nitric oxide and prostaglandins in fibroblasts (47), release of histamine from mast cells (48), and release of
substance P and neurokinin A from sensory neurons (49), processes that
can be considered anti-fibrotic. Our data demonstrate that activation
of the B2 receptor does not stimulate collagen or CTGF synthesis.
However, activation of the B1 receptor by
des-Arg10-kallidin stimulates type I collagen protein
synthesis, increases 1(I) collagen mRNA, and stabilizes CTGF
mRNA. TGF- and now des-Arg10-kallidin are the only
known stimulators of CTGF production.
The rate of mRNA degradation is determined, for the most part, by
the activity of destabilizing sequences, although stabilizing sequences
have been reported (50). Destabilizing sequences have been found in the
5'-untranslated region (51) and in the coding sequences (52). However,
cytokine mRNA degradation is mediated through increased exonuclease
activity, which appears to be regulated through adenine/uridine-rich
elements found in the 3'-untranslated region (53). GM-CSF mRNA
stabilization is well characterized. The GM-CSF 3'-untranslated region
contains several elements consisting of core AUUUA pentamers. Proteins
bind to AUUUA pentamers and appear to recruit other proteins, resulting
in increased exonuclease activity. Inhibition of protein synthesis by
CHX induces an increase in GM-CSF mRNA stability, presumably by
inhibiting synthesis of destabilizing proteins (54).
The CTGF 3'-untranslated region (996 nucleotides) contains three AUUUA
pentamers that potentially bind proteins involved in message
destabilization. Each AUUUA pentamer forms the core for nonamers that
are highly homologous to UUAUUU(U/A)(U/A), which is reported to
destabilize mRNA (55).
The mechanism that induces stabilization of GM-CSF mRNA appears to
be distinct from the mechanism that stabilizes CTGF mRNA. In the
studies presented here, CHX induces an increase in CTGF mRNA levels
at 6 h. The CHX-induced increase in CTGF mRNA is not sensitive
to inhibition of transcription by actinomycin D (data not shown),
suggesting that CHX induces an increase in CTGF mRNA stability by
inhibiting synthesis of destabilizing proteins. CHX could inhibit
specific RNases; however, GAPDH mRNA was not altered by CHX.
Co-stimulation with CHX and des-Arg10-kallidin further
increased the amount of CTGF mRNA, suggesting that the mechanisms
by which CHX and des-Arg10-kallidin induced an increase in
CTGF mRNA are distinct. Furthermore, TPA differentially regulates
the stabilization of CTGF mRNA and GM-CSF mRNA. Human lung
fibroblasts (WI38) exposed to TPA for 10 min exhibit increased GM-CSF
mRNA stability (44). In our system, TPA did not stabilize CTGF
mRNA. In fact, TPA attenuated the B1 receptor-mediated
stabilization of CTGF mRNA. It is possible that the divergent TPA
effects are cell type-specific.
The data presented are consistent with the interpretation that CTGF
mRNA is destabilized through labile destabilizing protein(s) that
binds to the AUUUA elements and that recruits RNases and other
cytoplasmic proteins. CHX blocks synthesis of the putative destabilizing protein(s), thus attenuating destabilization.
Des-Arg10-kallidin induces stabilization of CTGF mRNA
through post-translational modifications of the existing destabilizing
proteins. The des-Arg10-kallidin-mediated
post-translational modifications may reduce the RNA binding affinity of
the destabilizing proteins or sterically hinder the complex formation
of destabilizing proteins with other cytosolic proteins. The net result
of stimulation with des-Arg10-kallidin is an increase in
the message stability.
Activation of the B2 receptor by BK and kallidin is an integral part of
the early events of wound repair, mediating such processes as edema,
pain perception, and release or synthesis of pro-inflammatory factors
(3). When the possible contribution of the B2 receptor to fibrogenesis
was examined, it was found that activation of the B2 receptor does not
stimulate an increase in type I collagen production (36). Now it
appears that the B1 receptor system plays a distinct role in wound
repair, causing a rise in cytosolic Ca2+, stabilizing CTGF
mRNA, stimulating an increase in 1(I) collagen mRNA, and
stimulating collagen production. The potential pathophysiological role
of the B1 receptor in the development of fibrosis is intriguing. The B1
receptor is up-regulated at sites of inflammation and appears to be
refractory to desensitization (1). These characteristics, together with
the pro-fibrotic effects reported here, suggest that activation of the
B1 receptor may contribute to the excess collagen deposition associated
with chronic inflammatory conditions such as asthma and pulmonary fibrosis.
 |
FOOTNOTES |
*
This work was supported in part by NHLBI Grant P50HL56386
from the National Institutes of Health and by the Veterans Affairs Medical Center Merit Review Research Program.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.
Supported by NIDDK Grant R03DK53538 from the National
Institutes of Health.
**
Boston Veterans Affairs Medical Center Research Enhancement Awards
Program Fellow.
§
To whom correspondence should be addressed: Pulmonary Center,
Boston University School of Medicine, 715 Albany St., R3, Boston, MA
02118-2394. Tel.: 617-638-4860; Fax: 617-536-8093l; E-mail: ricupero@ bu.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
BK, bradykinin;
TGF- , transforming growth factor ;
CTGF, connective tissue growth
factor;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
FBS, fetal bovine serum;
DMEM, Dulbecco's modified Eagle's medium;
MLEC, mink lung epithelial cell;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
GM-CSF, granulocyte-macrophage colony-stimulating
factor;
CHX, cycloheximide.
 |
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S. C. Christiansen, J. Eddleston, K. M. Woessner, S. S. Chambers, R. Ye, Z. K. Pan, and B. L. Zuraw
Up-Regulation of Functional Kinin B1 Receptors in Allergic Airway Inflammation
J. Immunol.,
August 15, 2002;
169(4):
2054 - 2060.
[Abstract]
[Full Text]
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J. HEUSINGER-RIBEIRO, M. EBERLEIN, N. A. WAHAB, and M. GOPPELT-STRUEBE
Expression of Connective Tissue Growth Factor in Human Renal Fibroblasts: Regulatory Roles of RhoA and cAMP
J. Am. Soc. Nephrol.,
September 1, 2001;
12(9):
1853 - 1861.
[Abstract]
[Full Text]
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S. KONDO, S. KAGAMI, H. KIDO, F. STRUTZ, G. A. MULLER, and Y. KURODA
Role of Mast Cell Tryptase in Renal Interstitial Fibrosis
J. Am. Soc. Nephrol.,
August 1, 2001;
12(8):
1668 - 1676.
[Abstract]
[Full Text]
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A. Hahn, J. Heusinger-Ribeiro, T. Lanz, S. Zenkel, and M. Goppelt-Struebe
Induction of Connective Tissue Growth Factor by Activation of Heptahelical Receptors. MODULATION BY Rho PROTEINS AND THE ACTIN CYTOSKELETON
J. Biol. Chem.,
November 22, 2000;
275(48):
37429 - 37435.
[Abstract]
[Full Text]
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K. Suzuma, K. Naruse, I. Suzuma, N. Takahara, K. Ueki, L. P. Aiello, and G. L. King
Vascular Endothelial Growth Factor Induces Expression of Connective Tissue Growth Factor via KDR, Flt1, and Phosphatidylinositol 3-Kinase-Akt-dependent Pathways in Retinal Vascular Cells
J. Biol. Chem.,
December 22, 2000;
275(52):
40725 - 40731.
[Abstract]
[Full Text]
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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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