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*
Cardiovascular Research Institute and Departments of
Medicine and
Surgery, University of California, San Francisco, CA 94143
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chymase, a serine protease released from secretory granules by
degranulating mast cells, converts progelatinase B to an enzymatically
active form. The current work shows that these cells also secrete
gelatinase A. Furthermore, gelatinases A and B both colocalize to
-chymase-expressing cells of canine airway, suggesting that normal
mast cells are a source of gelatinases in the lung. In BR cells,
gelatinase B and -chymase expression are regulated, whereas
gelatinase A expression is constitutive. Progelatinase B mRNA and
enzyme expression are strongly induced by the critical mast cell growth
factor, kit ligand, which is produced by fibroblasts and
other stromal cells. Induction of progelatinase B is blocked by
U-73122, Ro31-8220, and thapsigargin, implicating phospholipase C,
protein kinase C, and Ca2+, respectively, in the
kit ligand effect. The profibrotic cytokine TGF-ß
virtually abolishes the gelatinase B mRNA signal and also attenuates
kit ligand-mediated induction of gelatinase B
expression, suggesting that an excess of TGF-ß in inflamed or injured
tissues may alter mast cell expression of gelatinase B, which is
implicated in extracellular matrix degradation, angiogenesis, and
apoptosis. In summary, these data provide the first evidence that
normal mast cells express gelatinases A and B and suggest pathways by
which their regulated expression by mast cells can influence matrix
remodeling and fibrosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TGF-ß (10),
lymphotoxin, and LPS (11) have all been shown to modulate gelatinase
expression in vitro. However, net enzymatic activity of MMPs in a
given milieu depends not only on their levels of expression, but
also on the relative coexpression of tissue inhibitors of
metalloproteinases (TIMPs) (9) and activator proteases that convert the
secreted pro-MMPs to active forms via proteolytic removal of the
propeptide domain (12, 13, 14, 15, 16, 17, 18).
Certain inflammatory disorders of the lungs, skin, and gut involve mast
cells, which degranulate to release stored mediators such as serine and
cysteine proteases, histamine, and heparin (19). Increased numbers of
degranulating mast cells in tissues with excessive collagen deposition
implicate them in matrix remodeling processes leading to fibrosis (20).
Coincident with mast cell hyperplasia are a paucity of other
inflammatory cells in the fibrotic areas and an abundance of
fibroblasts in close proximity to mast cells. While specific
contributions of mast cells to fibrotic processes remain unclear, these
observations suggest that mast cells may act independently or depend on
interactions with collagen-secreting fibroblasts (21). Both cell types
secrete factors that influence each other’s migration, proliferation,
differentiation, and survival. Fibroblasts secrete kit
ligand (KL, stem cell factor) that binds to Kit, a mast cell receptor
tyrosine kinase, whose ligation initiates a cascade of intracellular
signaling events regulating these mast cell functions (22, 23, 24). We
previously reported that BR dog mastocytoma cells constitutively
secrete progelatinase B, which is activated upon cleavage by
-chymase, a mast cell secretory granule-associated chymotryptic
serine protease (25, 26). Additional data demonstrated that incubation
of mastocytoma cells with phorbol ester increases expression of
gelatinase B while decreasing that of -chymase, predicting that
regulation of expression of these proteases is linked (26, 27). In the
present work, we demonstrate that resident mast cells in tissues
constitutively produce gelatinase B and that expression of gelatinase B
by cultured mast cells is regulated by not only c-kit
ligand, but also TGF-ß, an inflammatory cytokine implicated in tissue
fibrosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dog BR mastocytoma cells were maintained in continuous suspension culture, as previously described (25), in DMEM-H16 supplemented with 2% bovine calf serum. Cells were harvested and washed in PBS, and then incubated in serum-supplemented or serum-free medium alone, in the presence of 2 mM PMSF, 25 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or 5 ng/ml TGF-ß1 (R&D Systems, Minneapolis, MN), or with various concentrations of recombinant canine KL (kindly provided by Dr. Keith Langley, Amgen, Thousand Oaks, CA), U73122, Ro31-8220, or EGTA (Calbiochem, La Jolla, CA) for different times at 37°C. The conditioned medium was harvested and centrifuged at 500 x g for 5 min to remove cells and debris; decanted supernatant was stored at -20°C.
Purification
Gelatinase A was partially purified from media conditioned by BR cells using a protocol similar to that previously used to purify mastocytoma gelatinase B (26). Crude media conditioned by BR cells were brought to a final concentration of 0.5 M NaCl and subjected to sequential chromatography using DEAE-cellulose and gelatin-agarose. Bound gelatinases were eluted with 10% DMSO in 50 mM Tris-HCl (pH 7.5), containing 1 M NaCl and 0.02% NaN3. The eluate was concentrated using Centricon-10 spin columns (Amicon, Beverly, MA) and stored at -20°C.
Zymography
Gelatinase activity was detected by gelatin zymography, as previously described (25). Briefly, aliquots of medium conditioned by cells under various conditions were subjected to electrophoresis in 10% polyacrylamide gels containing gelatin (1 mg/ml). Gels were washed in 2.5% Triton X-100 to permit renaturation of gelatinases and stained with Coomassie blue after overnight incubation. Destaining visualized clear zones of lysis against a blue background, indicating gelatinase activity (7).
Development of -chymase Ig
-Chymase was purified from BR dog mastocytoma cells, as
previously described (28). Polyclonal anti--chymase Ig was
raised by injection of the purified -chymase into rabbits. IgG was
purified from antisera produced by Caltag Laboratories (South San
Francisco, CA) using Affigel A (Bio-Rad, Hercules, CA), according to
the manufacturer’s protocol.
Immunoblotting
Aliquots of partially purified mastocytoma gelatinase A,
purified recombinant human gelatinase A (Chemicon International,
Temecula, CA), sonicated cell lysates, purified dog -chymase (28),
or purified dog tryptase (29) were electrophoresed on a 10%
SDS-polyacrylamide gel and blotted onto PVDF membrane. The membrane was
washed in 10 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.3%
Tween-20 (TTBS) and then incubated with polyclonal sheep anti-human
gelatinase A (The Binding Site, San Diego, CA) or polyclonal rabbit
anti--chymase for 1 h. After washing in TTBS, the membrane
was incubated with goat anti-rabbit IgG alkaline phosphatase
conjugate (Sigma, St. Louis, MO) for 1 h. The membrane was washed
twice in TTBS, and then in 10 mM Tris-HCl (pH 7.5) containing 150 mM
NaCl. Immunoreactive protein was visualized by standard colorimetric
protocols using Fast Red TR/Naphthol AS-MX (Sigma), according to the
manufacturer’s protocols.
Immunohistochemistry
Lung tissues were obtained from normal euthanized dogs, sacrificed as part of experimental protocols in unrelated projects, via a tissue sharing program at the University of California, San Francisco. Tissues were washed in PBS (pH 7.40), fixed in PBS containing 4% paraformaldehyde for 1 h, and incubated in PBS containing 30% sucrose for 18 h at 4°C. Specimens were washed in PBS before freezing in Tissue-Tek OCT compound (Miles, Elkhart, IN) at -70°C.
To identify mast cells by metachromatic staining of sulfated proteoglycans in secretory granules, 5-µm-thick frozen airway sections were washed in PBS and incubated in 10 mM Tris-HCl (pH 8) buffer containing 5 mM magnesium acetate, 0.1 mM EDTA, and 0.1% methylene blue (w/v) for various periods of time. Tissue sections were washed in PBS, dehydrated in graded ethanols, cleared in xylenes, and mounted with a coverslip.
To detect mast cell proteases, sections were washed in PBS and blocked
for 10 min at 18°C in PBS containing 5% dehydrated milk, 3%
nonimmune goat serum, 0.1% Triton X-100, and 1% glycine. Sections
were washed in PBS and incubated for 18 h at 4°C with Abs
diluted in PBS containing 0.05% Tween-20. Sections were incubated
alone or with combinations of rabbit anti-dog -chymase or
commercially obtained sheep anti-human gelatinase A or sheep
anti-human gelatinase B (The Binding Site; sheep polyclonal Abs are
specific for either gelatinase A or B and do not cross-react with other
MMPs or Ags in the manufacturer’s quality control assays). Sections
were washed and incubated with either Texas Red-conjugated goat
anti-rabbit IgG (Vector Laboratories, Burlingame, CA) or
FITC-conjugated donkey anti-sheep IgG (Sigma) for 10 min at 18°C.
Slides were washed in PBS containing 0.05% Tween-20 and coverslipped
after treatment of sections with Vectashield Antifade reagent (Vector
Laboratories, Burlingame, CA). To colocalize either gelatinase A or
gelatinase B to -chymase-containing mast cells, sections were
incubated simultaneously with the appropriate primary Abs, followed by
simultaneous incubation with Texas Red- and FITC-conjugated secondary
Abs. Control experiments were performed as indicated to detect signals
resulting from recognition of nonspecific Ags by the secondary Abs.
Immunofluorescence signals were detected and photographed with a
fluorescence microscope.
RNA blotting
Poly(A)+ RNA was isolated from BR cells incubated
alone or with various combinations of TPA, KL, and TGF-ß using the
Poly(A)+ RNA MicroFast Track extraction kit (Invitrogen,
Carlsbad, CA). RNA blotting was performed as previously described (26).
Following size fractionation of poly(A)+ RNA and transfer
to Nytran Plus nylon membrane, the membrane was prehybridized for
2 h at 42°C in 50% formamide containing 5x Denhardt’s reagent
(1 g/L polyvinylpyrolidone, Ficoll, and BSA) and 5x SSPE (0.75 M NaCl,
50 mM NaH2PO4, and 5 mM EDTA) with 0.1% SDS
and 150 µg/ml salmon sperm DNA. The 2.3-kb dog gelatinase B cDNA
(26), the 2.2-kb human gelatinase A cDNA (American Tissue Type
Collection, Manassas, VA), or the 747-bp dog -chymase cDNA (30) was
random-prime labeled with [-32P]ATP. Labeled probes
were individually hybridized to the filter at 42°C overnight. Filters
were washed twice in 6x SSPE with 0.1% SDS at room temperature for 15
min, twice in 1x SSPE with 0.1% SDS at 37°C for 15 min, and once in
0.1x SSPE with 0.1% SDS at 55°C for 15 min. To remove previously
bound probe, blots were incubated in 5 mm Tris (pH 8), 0.2 mM EDTA,
0.05% pyrophosphate, and 0.1x Denhardt’s reagent at 65°C for
5 h. Densitometric data were obtained by analysis of
autoradiographic signals generated by hybridizing the blot with a
labeled probe. To account for possible variations in signal intensity
due to differing concentrations of total mRNA present in each lane, the
blot was also hybridized with a labeled probe for 
-actin.
Densitometric data were then compared with control values obtained with
the -actin probe.
Statistical analysis
Differences with a p value of <0.05 using Student’s two-tailed t test were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The gelatin zymogram depicted in Fig. 1
A reveals the presence of two
major bands (bands 1 and 2 at 
92 and 84 kDa, respectively) and
one minor band (band 3 at 66 kDa) of gelatinolytic activity in
medium conditioned by dog BR mastocytoma cells, as seen in lane
1. Incubation of cells with PMSF abolishes activity of band 2,
revealing a serine protease-dependent mechanism for its generation. As
revealed in our prior work (25), bands 1 and 2 represent gelatinolytic
activity of the zymogen and active forms of gelatinase B, respectively,
with extracellular proteolytic processing mediated by -chymase
released either by spontaneous degranulation or death of cultured
cells. Activity of bands 2 and 3 is ablated by Zn2+ or
Ca2+ chelators (data not shown), indicating that both are
MMPs. However, band 3, unlike band 2, is generated by BR cells even in
the presence of PMSF.
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62 kDa, as seen in lane 1 of Fig. 1
B. By
contrast, immunoreactive bands at 72 and 66 kDa are seen in
lane 2, which contains purified human gelatinase A. Work by
other investigators (31) has shown that purified dog progelatinase A
(which is identical in 13 of 15 N-terminal residues to human
progelatinase A) also yields a 62-kDa band by immunoblot analysis.
The basis for the differences in the electrophoretic migration of dog
and human progelatinase A is unclear. Thus, BR dog mastocytoma cells,
an extensively characterized mast cell model (25, 26, 27, 28, 29, 30, 32),
constitutively secrete progelatinase A. Tissue mast cell gelatinase expression
To perform cellular coimmunolocalization studies of tissue mast
cell proteases, polyclonal anti--chymase Ig was purified from
rabbit antisera. Purified anti--chymase Ig detects a 30-kDa
purified -chymase band and a band of similar size in BR cell
lysates, but it does not detect tryptase, the other major mast cell
serine protease (data not shown). To determine whether tissue mast
cells express gelatinases A and B, we performed immunohistochemistry on
sections of normal dog airway. Metachromatically staining mast cells in
the lung reside predominantly in bronchial airway subepithelium (33),
as shown in Fig. 2A.
Immunohistochemical analysis localizes gelatinase B and gelatinase A
signals to individual subepithelial cells, as shown in Fig. 2, D and G, respectively. (Immunoreactivity
localized to the epithelium in Fig. 2D results partially
from nonspecific binding of FITC anti-sheep IgG to dog tissues that
is not enhanced by nonimmune sheep or rabbit serum; similar background
signals are generated by Texas Red anti-rabbit IgG in the presence
of sheep or rabbit nonimmune serum. Alternatively, epithelial
immunoreactivity in Fig. 2, D and G, may also
represent signals for gelatinases in airway epithelial cells, as
previously described in vitro (34).) Detection of -chymase, a
secretory granule-associated serine protease that cleaves and activates
progelatinase B (25, 26), unequivocally reveals mast cells.
Immunofluorescence colocalization of signals for either gelatinase B
(Fig. 2E) or gelatinase A (Fig. 2H) to
-chymase-positive subepithelial cells (as seen in Fig. 2, F and I) establishes that -chymase-containing
mast cells express gelatinases in vivo. Thus, like other cells resident
in or recruited to the airways, mast cells may contribute gelatinases A
and/or B to the local milieu. These data substantiate our prior work,
which showed that mastocytoma cells secrete both progelatinase B and
-chymase (25), and support the notion that -chymase-dependent
progelatinase B activation may be a mechanism whereby tissue mast cells
acting independently can influence local gelatinase B activity.
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The importance of KL in mast cell migration, proliferation, and
survival suggested its candidacy as a regulator of gelatinase B
expression. Expression of Kit, the receptor for KL, is restricted
primarily to mast cells in connective tissue with limited expression in
skin melanocytes and intestinal interstitial cells of Cajal (24). As
seen in Fig. 3A, KL increases
levels of mast cell gelatinase B, although to a lesser extent than
those induced by phorbol. Levels of mast cell gelatinase B increase in
a dose-dependent fashion, with maximal induction at concentrations of
50–100 ng/ml, as shown in Fig. 3B. As demonstrated in Fig. 3C, addition of KL to cells results in induction of
gelatinase B expression, with increased levels noted at 4 h
compared with those of controls.
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Although gelatinases A and B share similar in vitro matrix
substrate specificities, wound injury models suggest that their
expression is temporally distinct and thus may be subject to regulation
by different extracellular signals. Zymographic analysis of medium
conditioned by cells shows that KL stimulation increases levels of
progelatinase B and its active, truncated forms, while the level of
progelatinase A remains unchanged, as seen in Fig. 4A. Autoradiograms and
densitometric data in Fig. 4, B and C, show that
KL increases the steady state level of gelatinase B mRNA by 5-fold,
although to a lesser extent than that induced by phorbol. By contrast,
neither KL nor phorbol alters steady state levels of gelatinase A mRNA.
These data suggest that KL:Kit interactions result in selective
induction of mast cell gelatinase B.
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-chymase expression by
KL
As shown in Fig. 5, incubation of
cells with KL decreases -chymase mRNA levels by 3-fold. Thus, KL
stimulation of cells results not only in selective up-regulation of the
expression of progelatinase B, but also divergent regulation of
-chymase. Whether decreased -chymase synthesis occurs remains
unclear.
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Ligation of Kit by KL alters expression of mast cell mediators by
initiating a cascade of intracellular signaling events (23). To explore
the role of early signal transduction pathways involved in KL-induced
mast cell gelatinase B expression, medium was harvested from cells
coincubated with KL and individual inhibitors selective for signal
transduction mediators, and analyzed by gelatin zymography. As seen in
Fig. 6A, treatment of cells
with increasing concentrations of U73122, an inhibitor of phospholipase
C activation, blocks KL-induced gelatinase B expression. Coincubation
with Ro31-8220, a bis-indoylmaleimide inhibitor selective for protein
kinase C, also blocks up-regulation of gelatinase B expression by KL,
as seen in Fig. 6B. As shown in Fig. 6C,
incubation of cells with KL in the presence of EGTA, a selective
nonmembrane-permeable Ca2+ chelator, also abolishes the
increase in gelatinase B induced by KL. Thus, these data suggest that
phospholipase C, protein kinase C, and an influx of extracellular
Ca2+ play a role in the induction of mast cell gelatinase B
expression by KL.
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Fibrotic tissue disorders demonstrate mast cell hyperplasia (20)
and increased levels of TGF-ß (35), an inflammatory cytokine that
antagonizes the effects of KL (36) and also modulates gelatinase B
expression (10, 37, 38). To explore the effect of TGF-ß on KL-induced
mast cell gelatinase B expression, medium conditioned by cells
incubated with combinations of KL and TGF-ß was analyzed by gelatin
zymography. As depicted in Fig. 7A, incubation of cells with
TGF-ß alone for 24 or 48 h (lanes 3 and
4) decreases gelatinase B levels. Preincubation of cells
with TGF-ß for 24 h also decreases the up-regulation of
gelatinase B expression following stimulation by KL (lane
5), an effect enhanced by prolonged incubation with TGF-ß for
48 h (lane 6). Whereas incubation with KL
increases the gelatinase B mRNA signal by 3-fold, treatment with
TGF-ß down-regulates expression of gelatinase B by 5-fold, as seen in
Fig. 7, B and C. Moreover, TGF-ß attenuates the
increase in the gelatinase B mRNA signal induced by KL. Thus, these
data suggest that ligation of TGF-ß to its receptor diminishes
KL-induced gelatinase B expression by stabilizing levels of the
protease’s mRNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chymase, a
secretory granule-associated serine protease exocytosed only in
response to an appropriate stimulus. The current work establishes that
tissue mast cells secrete both gelatinases A and B, and defines the
role of KL (a fibroblast mediator that binds to Kit) and the
profibrotic cytokine, TGF-ß, in mast cell gelatinase B expression.
Colocalization of gelatinases to -chymase-expressing cells in normal
dog airway tissues provides new insights into mast cell biology.
Immunohistochemical identification of mast cell gelatinase A and
gelatinase B expression in airway mast cells establishes that
production of these MMPs is not restricted to the malignant phenotype
of cultured mastocytoma cells. Whether all mast cells
express gelatinases and whether preformed gelatinases exist in the
cytosol or are sequestered in specific granules remain to be
determined. Expression of both progelatinase B and -chymase by
individual mast cells is consistent with our hypothesis that exocytosed
-chymase is an important activator of progelatinase B secreted
either by mast cells or by other cells in their vicinity (25). The
activation mechanism of mast cell progelatinase A, shown in other cell
types to be dependent on a trimolecular complex of the proform with
TIMP-2 and membrane-type MMP-1 (39), remains to be determined. Other
investigators have identified collagenase (MMP-1) and stromelysin
(MMP-3) in rodent and murine mast cells, respectively (40, 41).
Collectively, these data suggest that resident mast cells may
contribute a variety of MMPs, which may not only degrade target
substrates in tissues, but may also participate in overlapping
proteolytic cascades favoring pro-MMP activation (13, 14, 42, 43). In
the lungs, the relative paucity of mast cells in comparison with
MMP-expressing epithelial, stromal, and inflammatory cells (5, 6, 7, 8, 9, 34)
in normal or injured tissues suggests that mast cells may not be the
major source of gelatinases. However, hyperplasia of mast cells in
matrix-remodeling disorders such as pulmonary fibrosis (20) predicts
that contributions of MMPs from mast cells may be significant during
tissue injury and repair.
The critical role of KL:Kit interactions in the regulation of mast cell proliferation, migration, and survival (44) suggested that KL might also modulate mast cell expression of the gelatinase class of MMPs. Our data demonstrate that KL induces mast cell gelatinase B expression in a dose-dependent and selective manner. By contrast, gelatinase A expression in response to KL remains unchanged, thus substantiating the noncoordinate expression of gelatinases in tissue injury models (3). The frequent apposition of mast cells to fibroblasts in fibrotic tissues (21) suggests that fibroblast-derived KL (24) may not only contribute to the development of mast cell hyperplasia, but may also up-regulate gelatinase B expression, whose roles in tissue injury and remodeling remain speculative. Expression of KL by skin keratinocytes (24), alveolar macrophages (45), and airway epithelial cells (46) suggests that additional sources of KL may influence mast cell numbers and activities in a tissue-specific manner. Thus, interaction of fibroblasts with mast cells via KL may be important in the regulation of tissue expression of gelatinase B.
Phorbol stimulation studies were predictive of the effect of KL on mast
cell expression of -chymase, a chymotryptic serine protease that
activates progelatinase B by cleaving its catalytic domain (26). Our
prior work demonstrated that phorbol stimulates divergent expression of
these proteases: a multifold increase in gelatinase B expression (26)
contrasts with almost complete down-regulation of -chymase
expression (27). KL’s effect is similar, but of lesser magnitude.
Biochemical and physiologic consequences of KL’s effect on mast cell
expression of gelatinase B and -chymase are difficult to predict
because much about progelatinase B activation and the roles of both
proteases in vivo remain unknown. Convergent regulation of these
proteases would have suggested that KL stimulation favors conversion of
progelatinase B to active, mature forms. By contrast, divergent
regulation predicts that a local accumulation of progelatinase B might
occur because less -chymase is available to activate it. Whether KL
alters production of -chymase by mature mast cells is unknown. Since
progelatinase B may also be activated indirectly by a proteolytic
cascade initiated by mast cell tryptase or stromelysin (MMP-3) (12, 47), decreased availability of -chymase may not necessarily result
in an excess of mast cell progelatinase B. Therefore, definitive
assessment of the regulatory influence of KL on mast cell participation
in matrix remodeling awaits further clarification of the
interdependence of the in vivo activities of gelatinase B and
-chymase.
KL influences differentiation of bone marrow precursors into mast cells with distinct phenotypes based on protease expression. However, tissue-dependent heterogeneity of mast cells suggests that factors in addition to KL also direct mast cell protease expression (48). Effector cells recruited to inflamed or injured tissues contribute soluble mediators that most likely alter normal cell-cell and cell-matrix communication. Of the myriad cytokines released, sustained production of TGF-ß has been shown to contribute to abnormal tissue remodeling that progresses to fibrosis (35). Our data demonstrate that coincubation with TGF-ß attenuates mast cell production of gelatinase B in response to KL stimulation. How TGF-ß suppresses KL-induced gelatinase B expression is unclear. Prior studies demonstrate that TGF-ß neither disrupts binding of KL to Kit, nor alters mast cell Kit expression (36). It is possible that TGF-ß may interfere in KL-initiated intracellular signal transduction via competition for downstream mediators such as mitogen-activated protein kinases (49, 50) or transcription factor AP-1 (51).
Relative concentrations of TGF-ß and KL may therefore be important
determinants of mast cell protease expression in tissues undergoing
repair. Availability of soluble TGF-ß depends on release of its
bound, latent complex from extracellular matrix and its subsequent
conversion from latent to active forms (52, 53, 54). TGF-ß may alter mast
cell numbers by suppressing the ability of KL to rescue mast cells from
apoptosis (36), a cellular process that may also be regulated by
gelatinase B (55). Since -chymase also releases matrix-bound latent
TGF-ß (56), a complex feedback loop may regulate the effects of KL on
mast cells in the presence of TGF-ß. Moreover, mast cells themselves
may secrete TGF-ß (32, 57). Thus, persistent production of TGF-ß
may drive fibrotic processes not only by stimulating stromal cell
collagen deposition (58, 59, 60), but also by altering mast cell numbers
and their expression of gelatinase B.
In summary, our results demonstrate that resident tissue mast cells express gelatinase B, and that its expression in cultured mast cells may be regulated by both KL and TGF-ß. The data suggest that KL:Kit interactions may regulate gelatinase B expression in a manner that is both mast cell and tissue specific. Perturbations in signaling mediated by KL due to excess TGF-ß in inflammatory processes may alter mast cell gelatinase B expression and contribute to the development of fibrosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kenneth C. Fang, Cardiovascular Research Institute, University of California, Box 0911, San Francisco, CA 94143-0911.
3 Abbreviations used in this paper: MMP, matrix metalloproteinase; KL, recombinant canine kit ligand; TIMP, tissue inhibitor of metalloproteinase; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Received for publication October 13, 1998. Accepted for publication February 16, 1999.
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-chymase activates progelatinase B by cleaving the Phe88-Gln89 and Phe91-Glu92 bonds of the catalytic domain. J. Biol. Chem. 272:25628.-chymase gene. J. Immunol. 159:4367.[Abstract]
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 G. W. Wong, P. S. Foster, S. Yasuda, J. C. Qi, S. Mahalingam, E. A. Mellor, G. Katsoulotos, L. Li, J. A. Boyce, S. A. Krilis, et al. Biochemical and Functional Characterization of Human Transmembrane Tryptase (TMT)/Tryptase gamma . TMT IS AN EXOCYTOSED MAST CELL PROTEASE THAT INDUCES AIRWAY HYPERRESPONSIVENESS IN VIVO VIA AN INTERLEUKIN-13/INTERLEUKIN-4 RECEPTOR alpha /SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION (STAT) 6-DEPENDENT PATHWAY J. Biol. Chem., October 25, 2002; 277(44): 41906 - 41915. [Abstract] [Full Text] [PDF]
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 N. G. Frangogiannis, S. Shimoni, S. M. Chang, G. Ren, K. Shan, C. Aggeli, M. J. Reardon, G. V. Letsou, R. Espada, M. Ramchandani, et al. Evidence for an Active Inflammatory Process in the Hibernating Human Myocardium Am. J. Pathol., April 1, 2002; 160(4): 1425 - 1433. [Abstract] [Full Text] [PDF]
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D. Baram, G. G. Vaday, P. Salamon, I. Drucker, R. Hershkoviz, and Y. A. Mekori Human Mast Cells Release Metalloproteinase-9 on Contact with Activated T Cells: Juxtacrine Regulation by TNF-{alpha} J. Immunol., October 1, 2001; 167(7): 4008 - 4016. [Abstract] [Full Text] [PDF]
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B. T. Frank, J. C. Rossall, G. H. Caughey, and K. C. Fang Mast Cell Tissue Inhibitor of Metalloproteinase-1 Is Cleaved and Inactivated Extracellularly by {{alpha}}-Chymase J. Immunol., February 15, 2001; 166(4): 2783 - 2792. [Abstract] [Full Text] [PDF]
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 R. O. Sanchez Mejia, B. K. Lam, and J. P. Arm Matrix-Associated Transforming Growth Factor-beta 1 Primes Mouse Bone Marrow-Derived Mast Cells for Increased High-Affinity Fc Receptor for Immunoglobulin E-Dependent Eicosanoid Biosynthesis Am. J. Respir. Cell Mol. Biol., May 1, 2000; 22(5): 557 - 565. [Abstract] [Full Text]
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P. J. Wolters, M. Laig-Webster, and G. H. Caughey Dipeptidyl Peptidase I Cleaves Matrix-Associated Proteins and Is Expressed Mainly by Mast Cells in Normal Dog Airways Am. J. Respir. Cell Mol. Biol., February 1, 2000; 22(2): 183 - 190. [Abstract] [Full Text]
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