In primary cultures of pulmonary artery fibroblasts, duodenase induced a concentration-dependent increase in [3H]thymidine incorporation with a maximal effect observed at 30 nM.. PAR1, P
Trang 1Proteolytic action of duodenase is required to induce DNA synthesis
in pulmonary artery fibroblasts
A role for phosphoinositide 3-kinase
Alan D Pemberton1, Tatyana S Zamolodchikova2, Cheryl L Scudamore3, Edwin R Chilvers4,
Hugh R P Miller1and Trevor R Walker5
1 Department of Veterinary Studies, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Edinburgh, UK; 2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia;3Department of Veterinary Pathology, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Edinburgh, UK;4Respiratory Medicine Unit, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, UK;
5
Rayne Laboratory, Respiratory Medicine Unit, University of Edinburgh Medical School, Edinburgh, UK
Duodenase is a 29-kDa serine endopeptidase that displays
selective trypsin- and chymotrypsin-like substrate specificity
This enzyme has been localized to epitheliocytes of Brunner’s
glands, and as described here, to mast cells within the
intestinal mucosa and lungworm-infected lung, implying an
important additional role in inflammation and tissue
remodelling In primary cultures of pulmonary artery
fibroblasts, duodenase induced a concentration-dependent
increase in [3H]thymidine incorporation with a maximal
effect observed at 30 nM Pretreating duodenase with
soy-bean trypsin inhibitor abolished DNA synthesis, confirming
that proteolytic activity was an essential requirement for this
response PAR1, PAR2 and PAR4 activating peptides were
unable to induce [3H]thymidine incorporation in pulmonary
artery fibroblasts Likewise, pretreatment of fibroblasts with
TNFa, known to up-regulate PAR2 expression in other
systems, and IL-1b, did not enhance the potential of
duodenase to induce DNA synthesis Furthermore,
duo-denase increased GTPcS binding to fibroblast membranes
indicating that a G-protein-coupled receptor may mediate the effects of duodenase Duodenase-induced DNA syn-thesis and GTPcS binding were both found to be inhibited
by pertussis toxin, implying a role for Gi/o Selective inhi-bitors of MEK1 (PD98059) and protein kinase C (GF109203X) only partially inhibited duodenase-induced DNA synthesis, but both wortmannin (100 nM) and LY294002 (10 lM) inhibited this response completely, indicating a key role for PtdIns 3-kinase Furthermore, duodenase induced a 2.3 ± 0.1-fold increase in PtdIns 3-kinase activity in p85 immunoprecipitates, which was sensitive to inhibition by wortmannin These results suggest that duodenase can induce pulmonary artery fibroblast DNA synthesis in a PtdIns 3-kinase-dependent manner via a G-protein-coupled receptor which is activated by a proteo-lytic mechanism
Keywords: duodenase; fibroblasts; phosphoinositide 3-kinase; protease-activated receptor
Duodenase is a serine endopeptidase, originally isolated
from bovine duodenum, with a dual trypsin-like and
chymotrypsin-like primary substrate specificity, i.e cleaving
the C-terminal to both basic and hydrophobic amino-acid
residues [1] The closely related enzyme, sheep mast cell
proteinase-1 (sMCP-1) is 85% identical at the amino-acid
level [2] and, due to close similarity of the primary substrate
binding region, has a strikingly similar cleavage specificity
[3] Duodenase was originally immunolocalized to epithelial
cells of Brunner’s glands within the duodenum, and was an
activator of enteropeptidase [4] Other studies employing
esterase staining have provided evidence for the expression
of an enzyme with trypsin-like properties, distinct from tryptase, in intestinal mucosal mast cells, and in the lung around bronchioles and within the alveolar septa [5] This is consistent with data in sheep showing that sMCP-1 is located to mucosal mast cells of the gastrointestinal tract, and around small bronchi and alveolar walls in the lung [6] One of the many identified effects of mast cell proteinases
is their ability to induce cellular proliferation For example, both human mast cell tryptase and sMCP-1 have been shown to be mitogenic for fibroblasts [7,8] In patients with chronic inflammatory lung disorders, significant accumula-tion of mast cells occurs within the lungs and is believed to underlie the generation of pulmonary fibrosis, involving proliferation of mesenchymal cells to form the basis of a fibrotic scar [9] Recruitment of mast cells and release of their proteinases may therefore play a central role in the initiation of a proliferative response following injury or inflammation within the lung
The field of proteinase-mediated cellular activation has expanded rapidly following the discovery that a-thrombin mediates its actions through a receptor which contains a Ôtethered-ligandÕ, with activation occurring consequent to
Correspondence to T R Walker, Rayne Laboratory, Respiratory
Medicine Unit, University of Edinburgh Medical School, Teviot
Place, Edinburgh EH8 9AG, UK Fax: + 131 6504384,
Tel.: + 131 6511320, E-mail: tw@srv1.med.ed.ac.uk
Abbreviations: PAR, protease-activated receptor; sMCP-1, sheep mast
cell proteinase-1; DMEM, Dulbecco’s modified Eagle’s medium;
TNFa, tumour necrosis factor a; PtdIns 3-kinase, phosphoinositide
3-kinase.
(Received 14 September 2001, revised 7 December 2001, accepted 19
December 2001)
Trang 2proteolytic cleavage of the N-terminal exodomain This
thrombin receptor has since been termed PAR1
(protease-activated receptor-1) and is known to mediate the actions
of thrombin on platelets and other cell types [10]
Subse-quently, RT-PCR and Northern analysis have identified
mRNA for three additional members of this receptors
family termed PAR2, PAR3 and PAR4 [11] Interestingly,
thrombin has now been demonstrated to cleave and activate
PAR1, PAR3 and PAR4 whereas trypsin and tryptase
activate PAR2 [12] Certain other proteases, including
chymotrypsin and cathepsin G, appear to ÔdisarmÕ PAR1 by
cleaving the exodomain of the receptor without inducing
activation and thus preventing activation by thrombin [13]
All four receptors have a classical heptahelical structure
within the plasma membrane and are known to couple to
both Gq/11and Gi/oand stimulate phosphoinositide
turn-over although their other potential downstream signalling
targets have not been fully established [12] In this study we
have investigated the ability of duodenase to induce DNA
synthesis in bovine pulmonary artery fibroblasts, attempting
to elucidate which PAR subtype and signalling pathways
may be involved in mediating this effect We also provide
evidence for an additional mast cell origin of duodenase,
which has important implications with regard to the
potential in vivo role of this enzyme
M A T E R I A L S A N D M E T H O D S
Purification of duodenase from bovine jejunum
The protocol used for the purification of duodenase from
bovine jejunum was identical to that currently used for the
isolation of sMCP-1 from ovine gastrointestinal tissue In
brief, fresh bovine jejunal tissue was finely chopped and then
homogenized with 3 vol of 20 mM Tris/HCl pH 7.5 (all
procedures were carried out on ice) After centrifugation
(30 000 g for 30 min) and repetition of the above low salt
wash step, the pellet was homogenized with 3 vol of 20 mM
Tris/HCl (pH 7.5), 0.4M NaCl, 0.1% (v/v) Brij 35
Following repeat centrifugation, the supernatant was
diluted with 20 mMTris/HCl (pH 7.5), 0.1% (v/v) Brij 35
to < 0.1M NaCl, centrifuged again, and loaded onto a
column containing CM–Sepharose FF (Pharmacia),
equi-librated with the buffer described above After elution with
a 0.1–0.5M NaCl gradient, fractions containing both
chymotrypsin-like and trypsin-like activity were pooled,
then rechromatographed twice on a Mono-S column
(Pharmacia) using 0.05–0.35M NaCl gradients in 20 mM
Tris/HCl (pH 7.5), 0.1% (v/v) Brij 35, and then 20 mM
sodium phosphate (pH 7.0), 0.1% (v/v) Brij 35 The final
purification step involved gel filtration (Superdex 75,
Phar-macia) in NaCl/Pi(pH 7.4) containing 0.1% (v/v) Brij 35
The identity of the product was confirmed by N-terminal
amino-acid sequence analysis (P Barker, Babraham
Insti-tute, Cambridge, UK), and by comparing its ability to
hydrolyse specific peptide substrates (in 0.1M Tris/HCl,
pH 8.0) with duodenase
Immunohistochemical localization of duodenase
in jejunum and lung
Samples of fresh bovine jejunum were fixed in 10% (v/v)
formalin and 4% (w/v) paraformaldehyde, and processed
into paraffin blocks Sections (4 lm thick) were stained using 0.1% (w/v) toluidine blue (pH 0.5), followed by eosin counterstain, and duodenase detected using rabbit anti-duodenase serum (1 : 400), rabbit anti-(sMCP-1) IgG (1.2 lgÆmL)1) or control rabbit serum (1 : 400) [14], using NaCl/Pi(0.5MNaCl) containing 0.5% (v/v) Tween 80 for blocking and antibody dilutions The secondary antibody was biotinylated goat anti-(rabbit IgG) Ig (1 : 400; Vector Laboratories), followed by treatment with avidin–horse radish peroxidase (Vectastain ABC kit, Vector Laborato-ries) and diaminobenzidine (DAB kit, Vector LaboratoLaborato-ries) Following immunostaining, sections were counterstained with 0.1% (w/v) Mayer’s hematoxylin (Sigma) Samples of lung parenchyma were obtained at postmortem from a cow infected with the lungworm Dictyocaulus viviparus and fixed
in 4% (v/v) paraformaldehyde in NaCl/Pi Sections (5 lm thick) were prepared and stained with toluidine blue, rabbit anti-duodenase serum and control rabbit serum, as described above
Isolation and culture of bovine pulmonary artery fibroblasts
Sections of proximal bovine pulmonary artery were obtained from the local abattoir and pulmonary artery fibroblasts isolated using a primary explant procedure [15] Cells were cultured in supplemented Dulbecco’s modified Eagle’s medium (DMEM) containing foetal bovine serum (10% v/v), penicillin/streptomycin (5 UÆmL)1 and
5 lgÆmL)1, respectively) and amphotericin B (2.5 lgÆmL)1) Cells from passages 3–10 were used for all experiments Cells were incubated in serum-free DMEM for 48 h prior to experimentation
Assessment of [3H]thymidine incorporation Pulmonary artery fibroblasts at 80% confluence were quiesced for 48 h prior to addition of mitogens as indicated The cells were then incubated for an additional 20 h, with [3H]thymidine (0.1 lCiÆmL)1) added 4 h prior to harvest-ing Cells were washed twice with ice-cold NaCl/Pi, twice with trichloroacetic acid (5% w/v), twice with ethanol and finally were solubilized with NaOH (0.3M) [3H]Thymidine incorporation was determined by liquid scintillation counting
[35S]GTPcS binding to pulmonary artery fibroblast membranes
Pulmonary artery fibroblasts were lysed in ice-cold buffer containing 10 mMTris/HCl pH 7.4, 5 mMEDTA, homo-genized using a Polytron tissue homogeniser for 2· 10 s on ice and centrifuged at 500 g for 10 min at 4°C to remove intact cells Supernatants containing cell membranes were centrifuged at 50 000 g for 10 min and pellets washed with the buffer described above; this washing procedure was repeated twice The protein content of each pellet was determined after resuspension in 20 mM Hepes (pH 7.4) using a Pierce BCA protein assay reagent and the protein concentration adjusted to 1 mgÆmL)1 Binding of [35S]GTPcS was carried out by the addition of cell membranes (10 lg) to binding buffer (100 lL) containing
20 mMHepes pH 7.4, 100 mMNaCl, 3 mMMgCl, 10 lM
Trang 3GDP with 0.2 nM[35S]GTPcS and incubating for 60 min at
4°C Bound radioactivity was determined by filtration of
membranes onto Whatman GF-B filters using a Brandell
Cell Harvester and counted by scintillation counting
Nonspecific binding was determined in the presence of
100 lMunlabelled GTPcS
Assay of immunoprecipitated PtdIns 3-kinase
Bovine pulmonary artery fibroblasts were exposed to
mitogens as detailed in the figure legends, and the
reactions were terminated by rapid aspiration of the
media followed by the addition of ice-cold lysis buffer
(50 mMHepes, pH 7.5, 150 mMNaCl, 10% v/v glycerol,
1% v/v Triton X-100, 1.5 mM MgCl2, 1 mM EGTA,
10 lgÆmL)1leupeptin, 10 lgÆmL)1aprotinin, 1 mM
phen-ylmethanesulfonyl fluoride, 200 lM Na3VO4, 10 mM
sodium pyrophosphate, 100 mM NaF) PtdIns 3-kinase
was immunoprecipitated using antibodies specific to the
p85a regulatory subunit of PtdIns 3-kinase complexed to
Pansorbin (Calbiochem, Nottingham, UK) PtdIns
3-kinase activity in immunoprecipitates was assayed as
described previously, using sonicated phosphtidylinositol/
phosphatidylserine (3 : 1, v/v, 0.2 mgÆmL)1) vesicles and
[c-32P]ATP (10 lCiÆpoint)1) as substrates [16]
32P-Labelled phosphoinositide 3-phosphate was then
separated and quantified by thin layer chromatography
using a solvent system containing chloroform/methanol/
ammonia/water (20 : 15 : 3 : 5, v/v/v/v) and
autoradiog-raphy; 32P incorporation was determined by liquid
scintillation counting
Ca2+measurements using Fura-2
Bovine pulmonary artery fibroblasts (P4-10) were grown to
confluence in supplemented DMEM as described above,
washed with NaCl/Pi, and gently harvested into a solution
containing BSA (0.2% w/v), glucose (0.1% w/v) and CaCl2
(1 mM) in NaCl/Pi (NaCl/Pi+) Following centrifugation,
the cells were washed twice in NaCl/Pi+and resuspended
in the same buffer at a concentration of 1.5· 106
cellsÆmL)1 The cells were then incubated for 1 h at
37°C with an equal volume of 4 lMFura-2 AM (Sigma)
in NaCl/Pi+, washed three times with NaCl/Pi+ and
resuspended at 1–2· 106cellsÆmL)1 The cell suspension
was allowed to equilibrate to room temperature for
30 min and 2 mL aliquots of cells then used for Ca2+
measurements over the following 2–3 h Measurements
were made in 1· 1 cm quartz cuvettes, equipped with a
magnetic stirrer, using a PerkinElmer LS 50B fluorimeter
with fast-filter accessory This allowed measurement of
emission at 510 nm for quasi-simultaneous excitation at
340 and 380 nm, for Fura2 bound and unbound to Ca2+,
respectively Additions of agonists (trypsin, thrombin,
duodenase, chymotrypsin and bradykinin) were made in
small volumes (5–20 lL) At the end of each experiment,
the maximum fluorescence was obtained by disrupting the
cells by addition of 10% (v/v) Triton X-100 (40 lL), and
minimum fluorescence then determined using 20 lL of
0.4MEGTA in 3MTris base Results were analysed, and
conversions to intracellular Ca2+concentration performed,
using software (PerkinElmer)
In vitro comparison of PAR2 peptide cleavage
by duodenase, tryptase and trypsin The peptide Gly-Pro-Asn-Ser-Lys-Gly-Arg-Ser-Leu-Ile-Gly-Arg-Leu-Asp-Thr-Pro corresponding to residues 5–20
of rat PAR2 [PAR2(5–20)] was synthesized (G Bloom-berg, University of Bristol, UK) The activities of bovine trypsin, human skin tryptase (stabilized with heparin, a gift from Axis Pharmaceuticals, San Francisco, USA) and bovine duodenase were first standardized against the substrate CBZ-Lys-thiobenzyl ester This was undertaken using suitably diluted enzyme (10 lL) added to a cuvette containing 170 lL of 0.1M Hepes (pH 7.5), 10 lL 5,5¢-dithiobis-(2-nitrobenzoic acid) (10 mMin dimethylsulf-oxide) and 10 lL of N-carbobenzyloxy-Lys-thiobenzyl ester (10 mM in dimethylsulfoxide ) Initial cleavage rates
at 405 nm were measured over 90 s at 23°C, and specific activities calculated, with 1 U of activity defined as the amount of enzyme required to produce an absorbance increase of 1.0 UÆmin)1 For each enzyme, incubations were in 0.05M Hepes (pH 7.5), 0.15M NaCl, containing rat PAR2(5–20) (0.475 mgÆmL)1), alanyl-tryptophan (in-ternal standard, 0.05 mgÆmL)1) and 0.13 U of enzyme (total assay volume 200 lL) Samples (30 lL) were removed at varying time-points, and reactions terminated
by the addition of 30 lL 10% acetic acid These samples were then chilled on ice, and frozen ()20 °C) prior to analysis Intact PAR2(5–20) and internal standard peak heights were quantified in samples following RP-HPLC (Jupiter C5 column, Phenomenex) using a water/acetonit-rile gradient containing 0.1% trifluoroacetic acid The ratio
of intact PAR2(5–20) to internal standard peak heights was plotted against time Fractions collected from some runs were subjected to mass spectrometry (I Davidson, University of Aberdeen, Scotland, UK)
Materials Anti-duodenase serum and affinity-purified anti-(sMCP-1) IgG were prepared as described previously [4,8] Anti-(p85 PtdIns 3-kinase) Ig was obtained from TCS Biologicals (Botolph Claydon, UK) and [c-32P]ATP from Amersham (Amersham) PAR activating peptides, Ser-Phe-Leu-Leu-Arg-Asn for PAR1 and Gly-Tyr-Pro-Gly-Lys-Phe for PAR4 were obtained from Bachem Ltd (Saffron Walden, Essex, UK) and Gly-Arg-Leu and Ser-Leu-Ile-Gly-Arg-Leu-NH2for PAR2 were supplied by G Bloom-berg (University of Bristol, UK) All other chemicals were
of the highest commercial quality
R E S U L T S
Identification of duodenase The N-terminal amino-acid sequence of the product isolated from jejunum (Ile-Ile-Gly-Gly-His-Glu-Ala-Lys-Pro-His-Ser-Arg-Pro-Tyr-Met-Ala-Phe-Leu-Leu-Phe) was iden-tical to that originally described for duodenase [1] The first
of two peptide substrates analysed, bee venom melittin, was cleaved preferentially at Lys7, with secondary cleavage at Lys23, as previously described for duodenase [17] Porcine angiotensinogen (1–14) was rapidly and specifically cleaved
Trang 4Fig 1 Histochemical detection of jejunal and lung mast cells and immunoperoxidase localization of duodenase in bovine intestine and lung Repre-sentative positively stained mast cells are indicated by large arrows Panels (a–e) show localization of duodenase in bovine jejenum In panel (a) mast cells surrounding crypts in the jejunal mucosa are toluidine blue (pH 0.5)-positive (counterstained with eosin) Anti-duodenase Ig staining is shown at low magnification in panel (b), with abundant staining of cells with morphology and distribution similar to that shown for toluidine blue panel (a) Higher magnification in panels (c–e), shows bovine jejenum stained with control rabbit serum, rabbit anti-duodenase Ig and rabbit anti-(sMCP-1) Ig, respectively A similar pattern of staining is seen with anti-anti-duodenase Ig and anti-(sMCP-1) Ig, and no staining is observed in the control Sections of a bronchiole from bovine lung infected with the lungworm Dictyocaulus viviparus are shown in panels (f–h) In panel (f), duodenase-positive cells are abundant in the granulomatous reaction around the bronchiole Panel (g) shows an adjacent section incubated with control serum Panel (h) shows toluidine blue and eosin staining of a section adjacent to (f) Note the similar distribution of mast cells
in (f) and (h) and the presence of numerous eosinophils (small arrows) in the parasitized lung In association with the accumulation of mast cells there is increased fibrosis (*) and smooth muscle hypertrophy (arrowhead) All of the tissues were fixed in 4% (v/v) paraformaldehyde.
Trang 5at Phe8, as has been shown for duodenase [4] Therefore, the
jejunal enzyme we purified was identified as duodenase, or a
highly similar variant of the enzyme
Immunolocalization of duodenase
Toluidine blue staining identified abundant spindle or
stellate-shaped mast cells in bovine jejunum samples These
cells were located principally in the lamina propria (Fig 1a)
and submucosa (not shown) Immunostaining of
paraform-aldehyde-fixed sections with rabbit anti-duodenase serum
and affinity-purified rabbit anti-(sMCP-1) IgG detected cells
only in the lamina propria These strongly staining cells
showed a similar distribution and morphology to those seen
with toluidine blue within the lamina propria (compare
Fig 1a with Fig 1b,d,e) The distribution of positive cells
after labelling with anti-duodenase Ig or anti-(sMCP-1) IgG
was very similar, and in neither instance was there any
labelling of submucosal tissues Occasional intraepithelial
cells were weakly labelled (Fig 1d), and the identity of these
toluidine blue negative cells was not confirmed Tissues fixed
in neutral buffered formalin showed negligible mast cell
staining by comparison, and control rabbit serum was
negative regardless of the fixation procedure (Fig 1c)
Lungworm-infected lung parenchyma showed the presence
of large numbers of eosinophils and toluidine blue-positive
mast cells An example of their distribution around a
bronchiole is shown in Fig 1h, in which fibrosis and smooth
muscle hyperplasia was also evident Numerous cells were
also labelled with duodenase antiserum around bronchioles
(Fig 1f) and within the alveolar septa (not shown) Their
size and distribution as observed in adjacent sections was
similar to that of toluidine blue-positive cells (compare
Fig 1f,h) Control rabbit serum gave no labelling (Fig 1g)
Duodenase induces DNA synthesis in pulmonary artery
fibroblasts
The effect of duodenase on DNA synthesis was assessed
using [3H]thymidine incorporation in bovine primary
pul-monary artery fibroblasts Treatment of cells for 24 h with duodenase induced a concentration-dependent increase in [3H]thymidine incorporation which was maximal at 30 nM, achieving a 5.5 ± 0.8-fold increase above control values (Fig 2A) Pretreatment of duodenase with soybean trypsin inhibitor (3 mgÆmL)1, 15 min), an effective inhibitor of this enzyme [1] was found to inhibit completely the ability of this enzyme to induce [3H]thymidine incorporation in pulmo-nary artery fibroblasts (Fig 2B), confirming that the proteolytic activity of duodenase is essential for induction
of DNA synthesis Importantly, treatment of cells with duodenase (30 nM) for 10 min followed by the addition of soybean trypsin inhibitor (3 mgÆmL)1, 15 min) induced [3H]thymidine incorporation to a similar extent as addition
of duodenase alone (Fig 2B), suggesting a rapid signalling mechanism Furthermore, conditioned media generated by this method was used to assess whether duodenase could cleave and release a cell surface molecule that could interact
Fig 2 Duodenase induces DNA synthesis in pulmonary artery
fibro-blasts (A) quiescent cells were treated with duodenase (3–100 n M ) as
indicated for 20 h prior to addition of [ 3 H]thymidine (0.1 lCiÆwell)1):
incorporation was assessed after 4 h as detailed in Materials and
methods (B) [3H]Thymidine incorporation tested in cells treated with
duodenase (duod, 30 n M ) which had been pretreated with or without
soybean trypsin inhibitor (+ STI, 0.2 mgÆmL)1) for 15 min To
examine a role for duodenase-induced release of a mitogenic factor and
generation of conditioned media, duodenase was added to cells for
10 min prior to addition of soybean trypsin inhibitor for 15 min,
media removed and replaced with fresh quiescent media (duod + STI
removed) This conditioned media was transferred to untreated cells
(cond media) and [ 3 H]thymidine incorporation assessed as before.
(C) [3H]Thymidine incorporation tested in cells treated with duodenase
(30 n M ), PAR1 activating peptide (Ser-Phe-Leu-Leu-Arg-Asn,
100 l M ), PAR2 activating peptide (a, Leu-Ile-Gly-Arg-Leu; b,
Ser-Leu-Ile-Gly-Arg-Leu-NH 2 ; both 100 l M ) or PAR4 activating peptide
(Gly-Tyr-Pro-Gly-Lys-Phe, 100 l M ) [3H]Thymidine incorporation
was assessed as detailed in Materials and methods Results are
expressed as mean ± SEM-fold increase over control cells from four
separate experiments, each performed in triplicate.
Trang 6with cell surface receptors or induce secretion of a bioactive
molecule to induce DNA synthesis In these experiments,
addition of conditioned media to pulmonary artery
fibro-blasts had no significant effect on [3H]thymidine
incorpor-ation above control levels (Fig 2B) Hence duodenase,
purified from bovine jejenum is mitogenic for bovine
pulmonary artery fibroblasts and this effect is dependent
on the direct proteolytic activity of this enzyme
As the PARs described to date are activated by cleavage
of trypsin-like primary specificity, and as duodenase, (like
sMCP-1, which is also mitogenic in this system [8]), has a
trypsin-like component, activating peptides selective for
PAR1, PAR2 and PAR4 were used to investigate whether
the mitogenic effect of duodenase was mediated via a
known PAR mechanism Surprisingly, all PAR peptides
were unable to induce [3H]thymidine incorporation in
pulmonary artery fibroblasts (Fig 2C) It should be noted
that two forms of the PAR2 activating peptide were
assessed, the free form and the amido form, neither of which
showed ability to induce DNA synthesis (Fig 2C) Lack of
activation by these peptides is unlikely to be a consequence
of species differences in receptor sequences as
Ser-Leu-Ile-Gly-Arg-Leu (PAR2 activating peptide, mouse-derived
sequence, 100 lM) was reported to mobilize Ca2+in bovine
coronary artery smooth muscle cells [18] The PAR1
activating peptide Ser-Phe-Leu-Leu-Arg-Asn
(human-derived sequence, 100 lM) activated phospholipase C in
bovine tracheal smooth muscle cells (T R Walker &
E R Chilvers, unpublished observations) Furthermore,
this PAR1 activating peptide (100 lM) was found to induce
aggregation of isolated bovine platelets similar to that
induced by thrombin (T R Walker, unpublished
observa-tions)
Degradation of PAR2 model peptide
To further assess the potential interaction between
duoden-ase and PAR2, the ability of this enzyme to cleave a PAR2
substrate was investigated Under the experimental
condi-tions used, the known PAR2 activators bovine trypsin and
human mast cell tryptase rapidly cleaved the model PAR2
substrate PAR2(5–20) (t½ ¼ 3.5 and 3.4 min,
respective-ly) One cleavage product was resolved by HPLC and
identified by mass spectrometry as
Ser-Leu-Ile-Gly-Arg-Leu-Asp-Thr-Pro (m/z ¼ 971) (the other product
Gly-Pro-Asn-Ser-Lys-Gly-Arg was not resolved under the
chromatographic conditions used) This confirms the
capacity of trypsin and tryptase to cleave at the appropriate
activation site However, PAR2(5–20) was cleaved much
more slowly by duodenase (t½ 1200 min) Moreover, the
cleavage mixture exhibited HPLC peaks corresponding
both to the activation product
(Ser-Leu-Ile-Gly-Arg-Leu-Asp-Thr-Pro) and to other unidentified products, suggesting
multiple sites of cleavage of this substrate
Together, these results support the hypothesis that
duodenase acts independently of the known trypsin/
tryptase-sensitive PAR2 receptor
Duodenase induces GTPcS binding in pulmonary artery
fibroblast membranes
To establish the mechanism of action of duodenase,
[35S]GTPcS binding to fibroblast membranes was used as
an index of G protein activation Duodenase (30 nM) induced a 57.0 ± 2.3% increase in guanine nucleotide binding to pulmonary artery fibroblast cell membranes compared to controls, suggesting that the effects of duodenase are indeed mediated through a G-protein-coupled receptor Pre-treatment of cells with pertussis toxin (100 ngÆmL)1, 18 h) prior to cell fractionation and membrane isolation inhibited [35S]GTPcS binding by 80.8 ± 10.3%, suggesting that the predominant G-protein mediating this signal is a member of the Gi/ofamily (Fig 3)
Intracellular signalling pathway underlying duodenase-stimulated fibroblast proliferation
In order to identify a role for a downstream signalling pathway that may mediate the effect of duodenase on pulmonary artery fibroblasts, we examined a number of diverse signalling pathways that have been implicated
in agonist-stimulated DNA synthesis in other cell systems Pulmonary artery fibroblasts preloaded with the
Ca2+-binding dye fura-2 were stimulated with duodenase and fluorescence analysed as an index of Ca2+mobilization
As demonstrated in Fig 4, duodenase at concentrations up
to 90 nM was unable to induce Ca2+ mobilization In addition, thrombin, trypsin and chymotrypsin were also unable to induce Ca2+mobilization However, addition of bradykinin (5 lM) to these cells induced a rapid Ca2+ transient indicating that these cells were responsive to activation through other G-protein-coupled receptors (Fig 4) As anticipated, this response to bradykinin could
be desensitized by prior exposure to the agonist (Fig 4) These results suggest that this group of proteases do not appear to cause acute Ca2+mobilization or influx in these cells Of note, addition of a PAR2-activating peptide or addition of thrombin, which will act through PAR1, PAR3 and PAR4, all had no effect on Ca2+mobilization (Fig 4) These results demonstrate that Ca2+ mobilization is unlikely to be involved in mediating cell growth in pulmonary artery fibroblasts
Wortmannin (100 nM) and LY294002 (10 lM), two structurally distinct and selective inhibitors of PtdIns 3-kinase, completely blocked duodenase-induced [3H]thymidine incorporation, suggesting a key role for PtdIns 3-kinase in this response (Fig 5) In contrast, PD98059, a MEK1 inhibitor, caused only a partial
Fig 3 Effect of duodenase on [ 35 S]GTPcS binding Pulmonary artery fibroblasts were untreated (open bars) or pretreated with pertussis toxin (PTX, 100 ngÆmL)1, 18 h, hatched bars) prior to cell lysis and membrane isolation [35S]GTPcS binding was carried out as detailed in the Methods section, results are expressed as mean fold increase above control ± SEM from three experiments performed in triplicate.
Trang 7inhibition of DNA synthesis, reducing the response to
duodenase by 54% ± 13% (Fig 5), implying that
acti-vation of MEK1 and its downstream effectors may have a
modulatory role in duodenase-stimulated responses
Pre-incubation of cells with the protein kinase C inhibitor
GF109203X, at a concentration previously shown to fully
inhibit protein kinase C activity [19] again resulted in only
a modest reduction in duodenase-stimulated [3
H]thymi-dine incorporation (29% ± 10% inhibition from
stimu-lated control values), indicating that, although required
for a full mitogenic response, protein kinase C activation
does not appear to be critical for the initiation of this
response Pretreatment of pulmonary artery fibroblasts for
18 h with pertussis toxin, which ADP-ribosylates the
a subunit of Giand Goresulting in blockade of G protein
activation, inhibited duodenase-induced [3H]thymidine
incorporation by 52 ± 2.5%, suggesting involvement of
Gi/Go in mediating this component of cell growth
(Fig 5) In subsequent experiments, duodenase (30 nM)
was found to activate p85a-associated PtdIns 3-kinase in
pulmonary artery fibroblasts by 2.28 ± 0.14- fold above
control values, and pretreatment of these cells with
wortmannin (100 n , 20 min) inhibited this activity to
below basal levels (Fig 6) In combination with the major inhibitory effects of wortmannin and LY294002 on duodenase-stimulated [3H]thymidine incorporation, these results indicate a key role for a G-protein-coupled receptor/PtdIns 3-kinase pathway in mediating duoden-ase-stimulated DNA synthesis
Effect of inflammatory cytokines on duodenase-induced DNA synthesis
Pretreatment of pulmonary artery fibroblasts with the cytokines IL-1b and TNFa was undertaken to elucidate whether the effect of duodenase on DNA synthesis in our model system could be augmented by factors which are released at a site of inflammation Furthermore, TNFa has been reported to increase PAR2 expression and hence would allow further insight into a potential role of PAR2
in mediating the effects of duodenase [20] Exposure of pulmonary artery fibroblasts to duodenase resulted in a
Fig 5 Effect of signalling inhibitors on duodenase-induced DNA syn-thesis Pulmonary artery fibroblasts were pretreated with wortmannin (100 n M , 20 min) or LY294002 (10 l M , 20 min), PD98059 (10 l M ,
30 min), GF109203X (1 l M , 5 min) or pertussis toxin (PTX,
100 ngÆmL)1, 18 h) prior to addition of duodenase (30 n M ) [ 3 H]Thymidine incorporation was assessed as indicated in Methods, results are expressed as percentage mean ± SEM relative to untreated cells stimulated with duodenase Results are from four independent experiments each performed in triplicate.
Fig 6 Duodenase activates PtdIns 3-kinase in pulmonary artery fibro-blasts Pulmonary artery fibroblasts were incubated in the presence (hatched bars) or absence (open bars) of wortmannin (100 n M ) for
20 min prior to addition of duodenase (30 n M , duod) Reactions were terminated and PtdIns 3-kinase activity was assayed in p85a immunoprecipitates as detailed in Materials and methods Results are expressed as mean c.p.m ± SEM from a single experiment performed
in quadruplicate, representative of two others with similar results.
Fig 4 Effect of duodenase on Ca 2+ mobilization Pulmonary artery
fibroblasts preloaded with Fura-2 were stimulated with agonists as
indicated Intracellular Ca 2+ was analysed and plotted over time as
indicated Traces are representative of three separate experiments
which all gave very similar results.
Trang 85.23 ± 0.47-fold increase in [3H]thymidine incorporation
above control levels (Table 1) While pretreatment of cells
with IL-1b (10 ngÆmL)1) alone for 24 h induced an
increase in DNA synthesis by 49 ± 12% (n ¼ 8,
p< 0.05), it also resulted in a significant inhibition of
duodenase-induced [3H]thymidine incorporation relative
to IL-1b-treated control cells (Table 1) In contrast,
pretreatment with TNFa (10 ngÆmL)1) alone reduced the
level of [3H]thymidine incorporation by 69 ± 3%
(n ¼ 12, p < 0.05) (Table 1) but had no significant
effect on the relative magnitude of DNA synthesis
induced by duodenase: 6.19 ± 1.03-fold increase above
control values, respectively (Table 1)
D I S C U S S I O N
Mast cells present within the intestinal mucosa of rodents
express subset-specific chymases which are thought to act as
part of the innate immune response against intestinal
nematodes by increasing epithelial permeability [21] Similar
mucosal-specific mast cell subsets also exist in the sheep and
goat intestine [21,22] and are typified by the expression of
sMCP-1 and goat mast cell proteinase-1, respectively
Expulsion of intestinal nematodes in the sheep is associated
with simultaneous release of sMCP-1 into the gut lumen
and circulation [23]
The immunolocalization of duodenase to bovine
intesti-nal mucosal mast cells described here would suggest that it
too belongs to the ruminant mucosal mast cell proteinase
family, which are notable for their dual chymase and
tryptase-like activities It was possible to isolate duodenase
from bovine jejunum using methodology identical to that
employed for the purification of sMCP-1 from
gastrointes-tinal tissues However, duodenase has previously been
localized only to the epithelial cells of Brunner’s glands
located in the duodenal wall [4] This suggests either that
duodenase is present in both cell types, or that each site
produces distinct enzymes that are nonetheless highly
similar structurally, functionally and immunologically
Lungworm infection in sheep is known to involve a
pronounced mastocytosis [24], and sMCP-1 is upregulated
in mast cells recruited to sites of allergic lung inflammation
[25] The current observation of abundant duodenase-positive mast cells in lungworm-infected bovine lung shows the potential for local duodenase release by mast cells recruited to inflammatory sites in the bovine lung and is consistent with a putative role in tissue modelling
In this study, we have shown that the similarity between duodenase and sMCP-1 extends to the stimulation of pulmonary artery fibroblasts, with both enzymes able to induce DNA synthesis over a similar concentration range
As soybean trypsin inhibitor was able to completely inhibit the duodenase effect, this demonstrates that the catalytic activity is essential for its action However, only a short exposure to duodenase is required to induce maximal DNA synthesis suggesting a rapid activation profile Conditioned media from duodenase and soybean trypsin inhibitor-treated cells had no mitogenic effect, implying that duodenase acts directly on fibroblasts and does not release
a mitogenic mediator from the cell or medium to act in an autocrine or paracrine manner Furthermore, duodenase induces [3H]thymidine incorporation preferentially in sub-confluent cell cultures suggesting that close cell–cell contact and intercellular activation is not a requirement for DNA synthesis
It is now recognized that the mitogenic effect of other proteases such as thrombin and trypsin are mediated by protease-activated receptors [12] These are a family of seven-transmembrane or heptahelical receptors, which cou-ple to heterotrimeric G-proteins to transduce their signal, and are activated by cleavage of an extracellular portion of the receptor close to the N-terminus, thus exposing a new N-terminus that interacts with, and activates the receptor Receptors identified to date are PAR1, PAR2, PAR3 and PAR4; each has a similar mechanism of action has a distinct sequence at its cleavage site As a consequence, synthetic peptides have been developed that mimic the newly exposed N-terminus and act as specific activators [12] However, no selective ligand for PAR3 exists implying that this receptor requires other structural interactions to achieve activation [26] Indeed, recent data suggest that PAR3 does not mediate signal transduction directly but instead acts as a cofactor for the cleavage and activation of PAR4 [27] Thrombin has been shown to cleave and activate PAR1, PAR3 and PAR4, whereas trypsin cleaves and activates PAR2 As duodenase is capable of cleaving certain substrates with trypsin-like primary specificity, we initially hypothesized that induction of DNA synthesis by duoden-ase is mediated through a PAR2 mechanism
Surprisingly, we could find no evidence to support the involvement of a classic PAR2 in mediating the mitogenic effects of duodenase, specifically: (a) the synthetic peptide Ser-Leu-Ile-Gly-Arg-Leu, which is specific for PAR2, was unable to induce [3H]thymidine incorporation in fibroblasts, and a similar lack of mimickery was evident for peptides specific for PAR1 and PAR4; and (b) duodenase cleaved the model PAR2 substrate more slowly than either trypsin or tryptase, and generated a very different array of peptides, suggesting that duodenase may cleave PAR2 at different sites Activation of PAR3 by duodenase seems unlikely, as this receptor has limited intrinsic signalling capacity [27] and
so far has only been found to be activated by thrombin [12] Schechter et al [28] have described the action of mast cell tryptase on keratinocytes, as acting through a subpopula-tion of PAR2 receptors, suggesting the existence of subtypes
Table 1 Effect of cytokines on duodenase-induced DNA synthesis.
Bovine pulmonary artery fibroblasts were assessed for [3H]thymidine
incorporation induced by duodenase (30 n M ), following pretreatment
for 24 h with TNFa (10 ngÆmL)1) or IL-1b (10 ngÆmL)1) as indicated.
The values quoted represent the ratio of [3H]thymidine incorporation
to the mean [ 3 H]thymidine incorporation for the corresponding
un-treated control wells in the same experiment Results are expressed as
mean ± SEM Results in parentheses are corrected for the effects of
cytokines on baseline cell growth, and are expressed as the ratio of
[ 3 H]thymidine incorporation in proteinase-treated wells to that in
control wells for each cytokine treatment.
Untreateda + TNF-a a + IL-1b b
Control 1.00 ± 0.03 0.31 ± 0.03 1.49 ± 0.12
Duodenase 5.23 ± 0.47 1.90 ± 0.32
(6.19 ± 1.03)
3.96 ± 0.57 (2.65 ± 0.38)*
a n ¼ 12, over three separate experiments b n ¼ 8, over two
sepa-rate experiments * p > 0.001.
Trang 9of this receptor In addition, it has been demonstrated that
regulation of intestinal ion transport in rat jejenum is
mediated by a PAR that, although similar in many respects
to PAR2, showed distinct and atypical orders of potency
when a range of peptide agonists were assessed [29] These
reports and the data from this study, in particular the
pertussis toxin-sensitivity of DNA synthesis induction and
the ability of duodenase to stimulate [35S]GTPcS binding to
pulmonary artery fibroblast membranes, would suggest that
the mitogenic action of duodenase is mediated via direct
interaction with a proteolytically activated Gi/o-coupled
receptor While the precise PAR subtype remains to be fully
identified, it may be an atypical PAR2 that is not activated
by existing classic PAR2 peptides To date, no bovine PAR
sequences have been published and analysis of cleavage sites
on these receptors may reveal species-specific activation
motifs that are distinct from those in mouse, rat and
humans and explain the lack of efficacy of current
PAR2-activating peptides in our model system
A number of signalling pathways and intermediates such
as Ca2+mobilization, the ERK pathway, PtdIns 3-kinase
and protein kinase C have all been identified as mediators of
proliferative signals in a variety of cell types In pulmonary
artery fibroblasts, duodenase, trypsin, chymotrypsin,
thrombin and PAR2 peptides were unable to mobilize
Ca2+from intracellular stores As duodenase induces DNA
synthesis for these cells, these data would appear to
dissociate mobilization of intracellular Ca2+from induction
of DNA synthesis, a situation very similar to that previously
demonstrated in bovine airway smooth muscle [19,30]
These results also parallel those described for the effects of
human tryptase on fibroblasts, where tryptase is mitogenic
for these cells but does not act via PAR2 or Ca2+
-dependent pathways [28] Employing a range of selective
inhibitors, we investigated the roles of PtdIns 3-kinase,
MEK1/ERK, protein kinase C and pertussis toxin-sensitive
G-proteins Only partial inhibition of DNA synthesis was
achieved with maximally effective concentrations of
PD98059 (MEK1 inhibitor) and GF109203X (protein
kinase C inhibitor) indicating that each of these pathways
has a modulatory rather than a mandatory role to play in
mediating the proliferative response In contrast, a recent
report has shown that tryptase induces DNA synthesis in
canine tracheal smooth muscle through an
ERK1/2-depen-dent mechanism, proliferation being inhibited completely by
PD98059 [31] Moreover, in pulmonary artery fibroblasts,
inhibition of PtdIns 3-kinase by wortmannin or LY294002
inhibited completely duodenase-induced [3H]thymidine
in-corporation This would suggest that activation of PtdIns
3-kinase is the key regulatory step in the proliferative pathway
and that each of the other pathways interacts with this
pathway with the magnitude of the cellular response
determined by the integrated sum of each of these
components Our data is supported by previous reports
demonstrating that thrombin acts in a PtdIns 3-kinase- and
p70s6k-dependent manner to induce DNA synthesis in
pulmonary artery fibroblasts [32] In addition, this report
noted that downregulation of protein kinase C partially
attenuated thrombin-induced p70s6k activation, which
would concur with our findings that inhibition of protein
kinase C results in partial inhibition of DNA synthesis
To date, identification of downstream signalling
path-ways for PARs have principally concentrated on PAR1 and
PAR2 PAR1 couples to members of the G12/13, Gqand Gi families, interacting with various signalling pathways including phospholipase Cb, adenylyl cyclase, PtdIns 3-kinase and nonreceptor tyrosine kinases such as Src [33] PAR2 activation is associated with MAPK activation, phospholipase C activation and Ca2+mobilization How-ever, trypsin-induced MAPK activation was reported to occur independently of PAR2 in bovine pulmonary artery fibroblasts [34]
Inflammatory cytokines have previously been shown to induce selective upregulation of PAR2 receptors without affecting the thrombin receptor in human umbilical vein endothelial cells [20] To determine whether the prolifera-tive response of pulmonary artery fibroblasts could be modulated under inflammatory conditions, cells were treated for 24 h with TNFa; this resulted in a reduction
in the level of [3H]thymidine incorporation under control conditions, but had no significant influence on the relative magnitude of this response to duodenase In contrast, pretreatment of cells with IL-1b resulted in significant inhibition of duodenase-induced DNA synthesis and an enhanced level of baseline [3H]thymidine incorporation under control conditions These results suggest that these cytokines cause the fibroblasts either to become refractory
to mitogens or to enter into S-phase more slowly over the time period examined It remains to be established whether chronic exposure to TNFa and IL-1b would result in a sensitization of these cells to mitogenic stimuli These results support further our hypothesis that duodenase is not acting via a classical PAR2
In summary, this study has demonstrated that duoden-ase induces DNA synthesis in pulmonary artery fibro-blasts and that this response may be mediated by an atypical PAR, either an isoform of PAR2 or an uniden-tified receptor It is important to recognize that the current study was undertaken in a fully homologous system, using a bovine serine protease and bovine pulmonary fibroblasts This would indicate that the proteolytic event and subsequent downstream signalling and functional responses we have described may be an important consequence of duodenase release from Brun-ner’s glands, or of mast cell activation in vivo Indeed, mast cell hyperplasia is known to be a prominent event in many forms of chronic inflammation in the lung such as cryptogenic fibrosing alveolitis, and fibroblast proliferation
is the most significant feature in the pathology of these clinical conditions [9] The precise nature and character-ization of the receptor that mediates the effects of duodenase requires further investigation
A C K N O W L E D G E M E N T S
This work was funded by the Norman Salvesen Emphysema Research Trust, the Wellcome Trust, and the National Asthma Campaign (UK).
We thank Dr John Huntley and Ms Anne Mackellar for providing the bovine lung sections and Dr Jeremy Brown for help in preparing Fig 1.
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