Thrombin Stimulates Synthesis of Type IV Collagen and Tissue Inhibitor of Metalloproteinases-1 by Cultured Human Mesangial Cells
MITSUAKI KAIZUKA*,
HIDEAKI YAMABE*,
HIROSHI OSAWA*,
KEN OKUMURA* and
NOBORU FUJIMOTO
*Second Department of Internal Medicine, Hirosaki University School of
Medicine, Hirosaki, Japan. Biopharmaceutical Department, Research Institute, Fuji Chemical
Industries, Ltd., Takaoka, Japan.
Correspondence to Dr. Hideaki Yamabe, Second Department of Internal Medicine,
Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036, Japan.
Phone: 81 172 395057; Fax: 81 172 359190; E-mail:
yamabe{at}cc.hirosaki-u.ac.jp
.
Abstract. Glomerular accumulation of extracellular matrix (ECM)is
the common pathologic feature following glomerular injury,and the alteration
in the synthesis and degradation of ECM maybe involved in the glomerular
accumulation of ECM. Glomerularfibrin formation occurs in various forms of
human and experimentalglomerulonephritis, and it may play an important role
in progressiveglomerular injury. Thrombin, a multifunctional serine
proteinasethat is generated at the site of vascular injury, has central
functionsin hemostasis and it also shows various biologic effects. Inthis
study, it is hypothesized that thrombin may alter the productionand the
degradation of type IV collagen, which is an importantcomponent of ECM in the
glomeruli. Human mesangial cells (HMC)were cultured, and the levels of type
IV collagen, tissue inhibitorof metalloproteinase-1 (TIMP-1), and matrix
metalloproteinase-2(MMP-2) in the culture supernatants were measured by
enzymeimmunoassay using specific antibodies. MMP-2 activity was also
evaluatedby zymography using polyacrylamide/sodium dodecyl sulfate
gel-containinggelatin. Thrombin increased the production of type IV collagen
andTIMP-1 in a dose- and time-dependent manner, but it did notincrease
MMP-2. Thrombin also stimulated the gene expressionsof the type IV collagen
and TIMP-1 in HMC in a dose- and time-dependentmanner. Thrombin treated with
diisopropylfluorophosphate, aserine proteinase inhibitor, did not show any of
these effects.Hirudin, a natural thrombin inhibitor, and anti-transforming
growthfactor-ß-neutralizing antibody inhibited the stimulatingeffect of
thrombin. These findings suggest that thrombin maycontribute to the excessive
accumulation of ECM and progressionof glomerulosclerosis through an increase
of type IV collagenproduction and a decreased matrix degradation presumably
viaa transforming growth factor-ß-dependent mechanism.
Glomerular accumulation of extracellular matrix (ECM) is thecommon
pathologic feature of glomerular injury, and the alterationin the synthesis
and degradation of ECM may be involved in theglomerular accumulation of ECM.
Glomerular ECM has been shownto consist of type IV collagen, laminin, heparan
sulfate proteoglycan,fibronectin, and other proteins. Glomerular deposition
of typeIV collagen has been reported in human glomerular diseases
(1,2,)
andexperimental models
(3,4).
Matrix metalloproteinase-2 (MMP-2)is an important proteolytic enzyme
demonstrated to degrade typeIV collagen in in vivo as well as in
vitro studies, and it isshown to be synthesized by mesangial cells
in vivo as well asin vitro
(5,6,7).
Mesangial cells also synthesize tissue inhibitorof metalloproteinase-1
(TIMP-1), which can inhibit MMP-2
(8,9).
Recentinvestigations have shown that an imbalance between matrix-degrading
metalloproteinasesand the inhibitors of these enzymes may contribute to the
developmentof glomerulosclerosis
(7,10).
Intraglomerular fibrin formation is commonly observed in varioustypes of
glomerulonephritis, and the coagulation process hasbeen suggested to
contribute to the progression of glomerulosclerosis
(11,12,13).
Thrombin,a multifunctional serine protease that is generated in the
coagulationprocess and at the site of vascular injury, has central functions
inhemostasis. It is well known that thrombin has various biologiceffects
besides its role in hemostasis. For example, it hasbeen reported that
thrombin stimulates the mitogenesis of culturedfibroblasts
(14,15),
lymphocytes (16), endothelial
cells (17),vascular smooth
muscle cells
(18,19),
mesangial cells
(20,21),
andglomerular epithelial cells
(22). It is reported that
thrombincan regulate the production of several components of the fibrinolytic
systemin various cultured cells
(22,23,24,25).
Thrombin may thereforehave an important role in the deterioration of renal
functionand progression of glomerulosclerosis. In the present study,we
examined whether thrombin could influence the productionof type IV collagen,
TIMP-1, and MMP-2 by cultured human mesangialcells (HMC).
HMC Culture
HMC derived from human fetal kidneys (16 and 18 wk gestation)were kindly
provided by Dr. M. R. Daha (University Hospitalof Leiden, The Netherlands).
The cells were cultured in Dulbecco'smodified Eagle's minimum essential
medium (Life TechnologiesLaboratories, Grand Island, NY) with 20%
heat-inactivated fetalcalf serum (Bioserum, Canterbury, Australia) at
37°C in5% CO2. The cells were identified as glomerular
mesangial cellsby standard criteria
(26), i.e., they
exhibited a typical stellatemorphology when subconfluent and typical hillocks
were seenat confluence. Staining for actin, myosin, and type IV collagenwas
positive by immunofluorescence, and the cells were negativefor cytokeratin
and factor VIII. The cells were used betweenpassages 6 and 9 for the
experiments.
Assay of Type IV Collagen, TIMP-1, and MMP-2
The concentrations of type IV collagen and TIMP-1 in culturesupernatants
were quantified by enzyme immunoassay (EIA) usingthe Panaassay IV-C and human
TIMP-1 assay kits (Daiichi KagakuYakuhin, Tokyo, Japan). MMP-2 was measured
by a one-step sandwichEIA, as described previously
(27). This assay useed a pair
ofmonoclonal antibodies raised against the precursor form of MMP-2(proMMP-2)
purified from the conditioned medium of interleukin-1-treatedhuman
skin fibroblasts (CCD-41SK; American Type Culture Collection[ATCC] no.
CRL1505) (28) and against a
synthetic peptide correspondingto the N-terminal domain (residues 17 to 35)
of proMMP-2. Thesensitivity of this EIA was 0.24 ng/ml, and linearity was
obtainedbetween 1.0 and 500 ng/ml.
HMC were cultured in 12-well plates (Falcon; Becton Dickinson,Franklin
Lakes, NJ) and grown to confluence, which means HMCcovered the entire plate.
Subsequently, the cells were washedwith Hanks' balanced salt solution (Life
Technologies) and culturedwith Dulbecco's modified Eagle's minimum essential
medium containing0.2% bovine serum albumin (Sigma, St. Louis, MO) for 24, 48,
or72 h. Then type IV collagen, TIMP-1, and MMP-2 in the cell supernatants
weremeasured. The cells in each well were lysed in 1N NaOH, andthe protein
content was measured by the method of Lowry et al.
(29),using bovine serum
albumin as a standard. The levels of typeIV collagen, TIMP-1, and MMP-2 in
culture supernatants wereexpressed as ng per µg of lysed HMC protein.
Effect of Thrombin on the Production of Type IV Collagen, TIMP-1, and
MMP-2
We measured type IV collagen, TIMP-1, and MMP-2 in the supernatantsof HMC
incubated with 0.5 to 5.0 U/ml -thrombin (Sigma) for72 h to determine
its dose-dependent effect. The dosage of thrombinwas adopted according to a
previous report (23). Time
effectof thrombin was also examined by incubating HMC with or without5.0
U/ml thrombin for 24, 48, and 72 h. We also examined theeffect of
diisopropylfluorophosphate (DFP; Wako Pure ChemicalIndustries, Osaka,
Japan)-treated thrombin (DFP-thrombin) onthe production of type IV collagen
and TIMP-1. DFP is a serineproteinase inhibitor, and DFP-thrombin has no
enzymatic activity.In addition, we examined the inhibitory effect of
recombinanthirudin (American Diagnostica, Greenwich, CT), a specific natural
thrombininhibitor. DFP-treated thrombin was prepared by reacting thrombin
(200U/ml) with 10 mM DFP at pH 7.2 for 1 h at 37°C, and thenwas dialyzed
overnight against 0.01 M phosphate-buffered salineat pH 7.4. HMC were
incubated for 72 h with 5.0 U/ml thrombin,5.0 U/ml DFP-thrombin, 5.0 U/ml
thrombin plus 10.0 U/ml hirudin,or 10.0 U/ml hirudin, and the levels of type
IV collagen andTIMP-1 in the cell supernatants were measured. Finally, we
examinedthe effect of anti-transforming growth factor-ß
(TGF-ß)-neutralizingantibody (King Brewing Co., Kakogawa, Japan), which
is a polyclonalrabbit antibody, on the production of type IV collagen and
TIMP-1.HMC were incubated for 72 h with 5.0 U/ml thrombin, 5.0 U/mlthrombin
plus 50 µg/ml anti-TGF-ß antibody, or 5.0U/ml thrombin plus 50
µg/ml normal rabbit IgG, and thelevels of type IV collagen and TIMP-1 in
the cell supernatantswere measured. The cytotoxicity by thrombin,
DFP-thrombin, orhirudin was evaluated by assessing lactate dehydrogenase
(LDH)release from HMC. HMC were incubated in 24-well plates for 72h with 5.0
U/ml thrombin, 5.0 U/ml DFP-thrombin, or 10.0 U/mlhirudin, and the amount of
LDH in the cell supernatants wasmeasured. After removing the cell
supernatants, HMC were lysedby mellitin (50 µg/ml, Sigma), and the amount
of LDH inthe cells was also measured. LDH was quantified by a colorimetric
methodusing the LDH assay kit (Sanassay LDH, Sankou Junyaku, Tokyo,Japan).
The levels of LDH in the supernatants for 3 wells were113.3 ± 5.8
Wroblewski units/ml in 5.0 U/ml thrombin,118.3 ± 10.4 U/ml in 5.0 U/ml
DFP-thrombin, 105.3 ±13.6 U/ml in 10.0 U/ml hirudinin, and 110.0
± 10.0 U/mlin control. There was no significant difference among LDH
levelsin the cell supernatants of these groups. There was also nosignificant
difference among LDH levels in the cells of thesegroups. So it was considered
that no cytotoxicity was observedby these agents that we used in this
experiment. It is wellknown that thrombin enhances the proliferation of
cultured mesangialcells
(20,21).
We estimated the proliferation of HMC after confluenceby a colorimetric assay
using Cell Titer 96TM (Promega, Madison,WI) and by counting cell number.
There was not a significantincrease of HMC incubated for 72 h with 5 U/ml
thrombin.
Zymography of HMC Culture Supernatants
Sodium dodecyl sulfate (SDS) substrate gels (7.5%) were preparedby
including gelatin (DIFCO Laboratories, Detroit, MI) in thestandard Laemmli
acrylamide polymerization mixture at a finalconcentration of 1 mg/ml. The
samples were solubilized in nonreducingsample buffer and electrophoresed
using 25 mA/gel. After electrophoresis,the gels were washed in cold 50 mM
Tris/HCl, pH 8.0, containing50 mM NaCl, 10 mM CaCl2, and 0.05%
Brij 35 (Sigma). The gelswere then incubated overnight in fresh Tris buffer
at 37°C.Thereafter, the gels were stained with Coomassie Blue and zones
oflysis were visualized. Prestained protein molecular weight standards
(BethesdaResearch Laboratories, Gaithersburg, MD) were used for designation
ofmolecular weight. For analytical SDS-polyacrylamide gel electrophoresis,
HMCwere incubated with 0.5 to 5.0 U/ml -thrombin for 72 h, and10
µl of cell supernatants were electrophoresed to examinethe molecular
weight of their proteinases.
Northern Blot Analysis
HMC were stimulated with -thrombin (0.5 U/ml or 5.0 U/ml) for3 or 6
h, and then harvested and stored in liquid nitrogen untiluse. Total RNA was
extracted from the frozen cells by a single-stepmethod using RNAzol B
(Biotecx Laboratories, Houston, TX). ThenRNA samples (15 µg/lane) were
subjected to electrophoresison 1% agarose gel containing 2.2% formaldehyde
and transferredonto nylon membranes (Hybond-N, Amersham, Buckinghamshire,
UnitedKingdom) by capillary blotting. Membranes were hybridized overnightat
65°C with cDNA probes in the hybridization buffer containing5x
saline-sodium phosphate-ethylenediaminetetra-acetic acid,5x Denhardt's
solution, and 0.5% SDS. The cDNA probes were labeledwith
(-32P) dCTP (3000 Ci/mmol; Dupont, Boston, MA) by random
primerextension (30). After
hybridization, the membranes were washedand exposed to Reflection film
(Dupont) at -80°C with anintensifying screen for 6 h to 3 d. The cDNA
probes for humantype IV collagen-2, human TIMP-1, and human
glyceraldehyde 3-phosphatedehydrogenase (GAPDH) were purchased from American
Type CultureCollection (Rockville, MD).
Statistical Analyses
All data are expressed as mean ± SD. Results were comparedusing
one-way factorial ANOVA followed by multiple comparisontests, and two-way
repeated-measures ANOVA.
The digested zone of 72 kD of mesangial cell proteinase, whichis thought
to be MMP-2, was demonstrated by 7.5% SDS-polyacrylamidegel electrophoresis
including gelatin, and thrombin did notincrease the MMP-2 activity. Even when
HMC were incubated with5.0 U/ml thrombin, no gelatinase of other molecular
weight wasobserved (Figure
1).
Figure 1. Zymography of human mesangial cell (HMC) supernatant in a 7.5% sodium
dodecyl sulfate-polyacrylamide gel containing 1 mg/dl gelatin as a substrate.
The gelatin digestion zones represent activated matrix metalloproteinase-2
(MMP-2). Thrombin did not stimulate MMP-2 enzyme activity after 72 h
incubation when compared with untreated control supernatant. Representative
data from one of two separate experiments are shown. Lane 1, molecular marker;
lane 2, control; lane 3, 0.5 U/ml thrombin; lane 4, 5.0 U/ml thrombin.
Changes in the concentrations of type IV collagen, TIMP-1, andMMP-2 in the
HMC culture supernatants after 72 h of incubationwith -thrombin are
shown in Figure 2. The mean
level of typeIV collagen in four wells was 0.054 ± 0.007 ng/µgcell
protein (thrombin 0 U/ml), 0.071 ± 0.006 ng/µg(thrombin 0.5 U/ml),
0.107 ± 0.022 ng/µg (thrombin2.0 U/ml), and 0.478 ± 0.049
ng/µg (thrombin 5.0U/ml) (Figure
2A). The mean level of TIMP-1 in four wells was15.50 ±
4.59 ng/µg cell protein (thrombin 0 U/ml),17.82 ± 4.57 ng/µg
(thrombin 0.5 U/ml), and 27.82± 1.45 ng/µg (thrombin 5.0 U/ml)
(Figure 2B). Themean level of
NMP-2 in four wells was 0.61 ± 0.12 ng/µgcell protein (thrombin 0
U/ml), 0.62 ± 0.16 ng/µg(thrombin 0.5 U/ml), and 0.56 ± 0.03
ng/µg (thrombin5.0 U/ml) (Figure
2C). Thrombin significantly increased theproduction of type IV
collagen and TIMP-1, but it did not changethe amount of NMP-2. Time effect of
thrombin was also examinedby incubating HMC with or without 5.0 U/ml thrombin
for 24,48, and 72 h. Thrombin significantly stimulated the productionof type
IV collagen (Figure 3A) and
TIMP-1 (Figure 3B) in a
time-dependentmanner. We also examined the effects of DFP-thrombin,
recombinanthirudin, and anti-TGF-ß-neutralizing antibody on the
productionof type IV collagen and TIMP-1. DFP-thrombin (5.0 U/ml) didnot
stimulate type IV collagen production, and hirudin (10.0U/ml) inhibited the
stimulating effect of thrombin, while hirudinitself did not influence type IV
collagen production (Figure
4A).Anti-TGF-ß antibody blocked the stimulating effect of
thrombinin type IV collagen production, while normal rabbit IgG didnot.
Anti-TGF-ß antibody itself did not influence typeIV collagen production
(Figure 4B). DFP-thrombin (5.0
U/ml)did not stimulate TIMP-1 production. Recombinant hirudin (10.0U/ml)
inhibited the stimulating effect of thrombin, while hirudinitself did not
influence TIMP-1 production (Figure
5A). Anti-TGF-ßantibody blocked the stimulating effect of
thrombin in TIMP-1production by HMC, while normal rabbit IgG did not.
Anti-TGF-ßantibody itself did not influence TIMP-1 production
(Figure 5B).
Figure 2. Thrombin stimulated the production of type IV collagen (A) and tissue
inhibitor of metalloproteinase-1 (TIMP-1) (B) by HMC, but it did not stimulate
MMP-2 production (C). Values are the mean ± SD for four wells, and
representative data from one of two experiments are shown, respectively.
*P < 0.02 versus 0 U/ml thrombin;
**P < 0.001 versus 0, 0.5, and 2.0 U/ml
thrombin; ***P < 0.02 versus 0 and 0.5 U/ml
thrombin.
Figure 3. Time course of the production of type IV collagen (A) and TIMP-1 (B) by HMC
in the absence ([UNK]) or presence () of 5 U/ml thrombin. Thrombin
stimulated the production of type IV collagen (P < 0.0001) and
TIMP-1 (P < 0.005) in a time-dependent manner. Results are
expressed as ng per µg cell protein. Values are the mean ± SD for
four wells.
Figure 4. Thrombin (T; 5 U/ml) stimulated type IV collagen production by mesangial
cells. However, 5 U/ml diisopropylfluorophosphate (DFP)-thrombin (DFP-T) did
not stimulate type IV collagen production. Hirudin (H; 10 U/ml) inhibited the
stimulating effect of thrombin, but hirudin itself did not influence type IV
collagen production. (B) Anti-transforming growth factor-ß (TGF-ß)
antibody blocked the stimulating effect of thrombin in type IV collagen
production by mesangial cells, while normal rabbit IgG (nl.Rb.IgG) did not.
Anti-TGF-ß antibody itself did not influence type IV collagen production.
Values are the mean ± SD for three wells, and representative data from
one of two separate experiments are shown, respectively. C, control.
*P < 0.0001 versus other groups;
**P < 0.0001 versus C and T +
anti-TGF-ß.
Figure 5. Thrombin (T; 5 U/ml) stimulated TIMP-1 production by mesangial cells.
However, 5 U/ml DFP-thrombin (DFP-T) did not stimulate TIMP-1 production.
Hirudin (H; 10 U/ml) inhibited the stimulating effect of thrombin, but hirudin
itself did not influence TIMP-1 production. (B) Anti-TGF-ß antibody
blocked the stimulating effect of thrombin in TIMP-1 production by mesangial
cells, while normal rabbit IgG (nl.Rb.IgG) did not. Anti-TGF-ß antibody
itself did not influence TIMP-1 production. Values are the mean ± SD
for three wells, and representative data from one of two separate experiments
are shown, respectively. C, control. *P < 0.0005
versus other groups; **P < 0.01
versus C and T + anti-TGF-ß.
Northern blot analysis showed that mesangial cells expressedtype IV
collagen and TIMP-1 mRNA under basal conditions. Thrombin(0.5 to 5.0 U/ml)
upregulated the gene expressions of type IVcollagen and TIMP-1 in a
dose-dependent manner compared withthe expression of glyceraldehyde
3-phosphate dehydrogenase (GAPDH),which is used as internal control
(Figure 6A), and incubation
with5.0 U/ml thrombin for 3 and 6 h upregulated the gene expressionsof type
IV collagen and TIMP-1 in a time-dependent manner
(Figure 6B).
Figure 6. (A) Type IV collagen mRNA and TIMP-1 mRNA expressions of HMC incubated with
media alone, media with 0.5 U/ml thrombin, and media with 5.0 U/ml thrombin
for 6 h. Type IV collagen mRNA and TIMP-1 mRNA of mesangial cells incubated
with thrombin were increased in a dose-dependent manner compared with GAPDH.
(B) Type IV collagen mRNA and TIMP-1 mRNA expressions of HMC before
incubation, 3 h, and 6 h after incubation with 5.0 U/ml thrombin. Type IV
collagen mRNA and TIMP-1 mRNA of mesangial cells incubated with thrombin were
increased in a time-dependent manner compared with GAPDH. Representative data
from one of two separate experiments are shown, respectively.
The accumulation of ECM is a common feature of glomerulosclerosisin many
glomerular diseases. Because type IV collagen is animportant component of the
glomerular basement membrane andthe mesangial matrix, alterations of its
synthesis and degradationmight contribute to the development of
glomerulosclerosis. Glomerularcells have the ability to produce several ECM
components, aswell as several metalloproteinases and TIMP
(10,31).
Metalloproteinasescan degrade the glomerular ECM and glomerular basement
membrane,while TIMP inhibit their activity. Thus, an imbalance in the
productionof these enzymes may induce the destruction or the impaired
remodelingof glomerular architecture
(7,8,9,32).
Recent investigationshave suggested a pathologic role of matrix-degrading
metalloproteinasesand their inhibitors in glomerular injury
(7,32,33).
The geneexpressions of these enzymes in the glomeruli are often alteredin
pathologic situations
(33).
The coagulation process, including platelet aggregation andlocalized
intravascular coagulation, has been implicated aseither a primary
pathogenetic factor or a secondary mediatorof glomerular injury in a variety
of glomerular and vascularrenal disease
(11,12,13).
Fibrin-like material has been demonstratedin the glomeruli, and fibrin
degradation products have beenfound in the serum and urine of patients with
glomerulonephritis.However, the mechanisms responsible for glomerular injury
bythe coagulation process have not been well established. Depressionof blood
flow caused by intraglomerular coagulation may injureglomerular cells and
coagulation factors and/or platelets maycause glomerular injury by altering
glomerular metabolism. Thrombinis the serine protease that converts
fibrinogen to fibrin, andis well known to have various biologic activities.
Thrombinis mitogenic for lymphocytes, endothelial cells, and other typesof
cells
(16,17,18,19),
and it can regulate the production ofcomponents of the fibrinolytic system by
various cultured cells
(22,23,24,25).
In the present study, we found that thrombin accelerated theproduction of
type IV collagen and TIMP-1, but not MMP-2, bycultured HMC. It was also found
that thrombin upregulated thegene expressions of type IV collagen and TIMP-1
in HMC. Suchchanges could lead to the excessive production and reduced
degradationof type IV collagen, and cause the glomerular accumulation oftype
IV collagen. In contrast, a stimulating effect was notseen in DFP-treated
thrombin. DFP is a serine proteinase inhibitorand it blocks the enzyme
activity of thrombin. The specificityof the effect of thrombin was also
tested by addition of hirudin,a natural specific thrombin inhibitor that
blocks both the proteolyticand membrane receptor binding functions of
thrombin
(34,35).
At10 U/ml, hirudin significantly inhibited the stimulating effectof thrombin
in the production of type IV collagen and TIMP-1.The enzymatic activity of
thrombin has been reported to be necessaryfor its mitogenic effect on smooth
muscle cells
(36,37),
whileinactivation of the thrombin catalytic site by both hirudinand DFP
blocks the thrombin-mediated increase of tissue plasminogenactivator and
plasminogen activator inhibitor-1
(23). The recentcloning of
the thrombin receptor on platelets and vascular endothelialcells has revealed
a novel proteolytic mechanism of receptoractivation
(38), in which thrombin
cleaves the receptor N-terminalextension site to create a new N-terminal that
functions asa tethered ligand and activates the receptor. This notion is
supportedby the fact that DFP-thrombin did not stimulate the productionof
type IV collagen and TIMP-1, and hirudin completely suppressedTIMP-1
production and it considerably suppressed type IV collagenproduction.
To our knowledge, there have been no previous investigationsregarding the
effects of thrombin on type IV collagen, MMP-2,and TIMP-1 production by
cultured cells. Thrombin is generatedat sites of glomerular injury by
activation of the intrinsicor extrinsic coagulation pathway. Our findings
suggest thatthrombin may contribute to glomerular sclerosis in part by
stimulatingECM production as well as by increasing metalloproteinase
inhibitorexpression in mesangial cells.
Many in vivo and in vitro studies have shown the effects
ofcytokines and growth factors on the accumulation of ECM
(39,40,41,42,43,44,45,46,47),
whichmay depend on the balance between synthesis and degradation.It is
reported that various modulators may influence the balanceof
metalloproteinases and TIMP within the mesangium and determinethe overall
direction of matrix metabolism
(48,49).
Kitamuraet al. demonstrated that a high glucose concentration
upregulatesthe gene expression of type IV collagen, TIMP, and laminin,but
not that of transin, which is a matrix metalloproteinase
(8).They also showed that
heparin selectively inhibits expressionof the transin gene in cultured
mesangial cells (50). Martin
etal. reported that HMC constitutively secrete MMP-2 and a small
amountof MMP-9, which is a 92-kD enzyme, and when the cells were stimulated
byIL-1ß or phorbol myristate acetate, MMP-9 was increasedin a
dose-dependent manner, while MMP-2 remained unaffected.They thought that
MMP-2 may have an important role in the regularturnover of matrix, while
MMP-9 may become predominant in pathologicsituations
(51). However, in our study,
even when HMC were incubatedwith 5.0 U/ml thrombin, the MMP-2 activity was
not increasedand no proteinase of other molecular weight was observed. Lovett
etal. demonstrated that low concentrations (0.1 to 1.0 ng/ml)of
TGF-ß increased the production of TIMP in culturedHMC, while high
concentrations of TGF-ß increased gelatinaseactivity
(52). TGF-ß has also been
shown to decrease theproduction of ECM-degrading proteinases and increase the
productionof their inhibitors in cultured human fibroblasts
(53,54).
TGF-ßhas been thought to play a central role in the accumulationof ECM
in glomerulonephritis
(55,56,57,58).
In our study, the stimulating effect of thrombin was completelyinhibited
by anti-TGF-ß antibody. Several authors havereported that thrombin can
modulate the production of some cytokines
(18,59).
Wehave reported that thrombin also stimulates TGF-ß productionin
cultured HMC (60). Taken
together, these data suggest thatthrombin stimulates the production of type
IV collagen and TIMP-1through a TGF-ß-dependent mechanism.
In summary, we have shown that thrombin could stimulate theproduction of
type IV collagen and TIMP-1 by HMC. This effectof thrombin was blocked with
anti-TGF-ß antibody. Thrombinis released when glomerular injury occurs,
and it may play animportant role via a TGF-ß-dependent mechanism in the
progressionof glomerulosclerosis.
Striker LM, Killen PD, Chi E, Striker GE: The composition of
glomerulonephritis. I. Studies in focal sclerosis, crescentic
glomerulonephritis and membranoproliferative glomerulonephritis.
Lab Invest 51:181
-192, 1984[Medline]
Fukatsu A, Matsuo S, Killen PD, Martin GR, Andres GA, Brentijens
JR: The glomerular distribution of type IV collagen and laminin in human
membranous glomerulonephritis. Hum Pathol19
: 64-68,1988[Medline]
Matsuo S, Brentijens JR, Andres G, Foidart JM, Martin GR,
Martinez-Hernandez A: Distribution of basement membrane antigens in glomeruli
of mice with autoimmune glomerulonephritis. Am J
Pathol 122:36
-49, 1986[Abstract]
Woessner JJ: Matrix metalloproteinases and their inhibitors in
connective tissue remodeling. FASEB J5
: 2145-2154,1991[Abstract]
Davies M, Thomas GJ, Martin J, Lovett DH: The purification and
characterization of a glomerular-basement membrane-degrading neutral
proteinase from rat mesangial cells. Biochem J251
: 419-425,1988[Medline]
Davies M, Martin J, Thomas GJ, Lovett DH: Proteinases and
glomerular matrix turnover. Kidney Int41
: 671-678,1992[Medline]
Kitamura M, Kitamura A, Mitarai T, Maruyama N, Nagasawa R, Kawamura
T, Yoshida H, Takahashi T, Sakai O: Gene expression of metalloproteinase and
its inhibitor in mesangial cells exposed to high glucose. Biochem
Biophys Res Commun 185:1048
-1054, 1992[Medline]
Carome MA, Striker LJ, Peten EP, Elliot SJ, Yang CW, Stetler S-W,
Reponen P, Tryggvason K, Striker GE: Assessment of 72-kilodalton gelatinase
and TIMP-1 gene expression in normal and sclerotic murine glomeruli.
J Am Soc Nephrol 5:1391
-1399, 1994[Abstract]
Baricos WH, Shah SV: Proteolytic enzymes as mediators of glomerular
injury [Editorial]. Kidney Int40
: 161-173,1991[Medline]
Deguchi F, Tomura S, Yoshiyama N, Takeuchi J: Intraglomerular
deposition of coagulation-fibrinolysis factors and a platelet membrane antigen
in various glomerular diseases. Nephron51
: 377-383,1989[Medline]
Ono T, Kanatsu K, Doi T, Sekita K, Onoe C, Nagai H, Muso E, Yoshida
H, Tamura T, Kawai C: Relationship of intraglomerular coagulation and platelet
aggregation to glomerular sclerosis. Nephron58
: 429-436,1991[Medline]
Pollak VE: Glomerular thrombosis predicts progression of
glomerulonephritis: Can we prevent progression? Am J Kidney
Dis 26: 535-540,1995[Medline]
Gordon EA, Carney DH: Thrombin receptor occupancy initiates cell
proliferation in the presence of phorbol myristate acetate. Biochem
Biophys Res Commun 141:650
-656, 1986[Medline]
Carney DH, Scott DL, Gordon EA, Labelle EF: Phosphoinositides in
mitogenesis: Neomycin inhibits thrombin-stimulated phosphoinositide turnover
and initiation of cell proliferation. Cell42
: 479-488,1985[Medline]
Chen LB, Teng NNH, Buchanan JM: Mitogenicity of thrombin and
surface alterations on mouse splenocytes. Exp Cell Res101
: 41-46,1976[Medline]
Herbert JM, Dupuy E, Laplace MC, Zini JM, Bar-Shavit R, Tobelem G:
Thrombin induces endothelial cell growth via both a proteolytic and a
non-proteolytic pathway. Biochem J303
: 227-231,1994
Weiss RH, Maduri M: The mitogenic effect of thrombin in vascular
smooth muscle cells is largely due to basic fibroblast growth factor.
J Biol Chem 15:5724
-5727, 1993
Kanthou C, Parry G, Wijelath E, Kakkar VV, Demoliou-Mason C:
Thrombin-induced proliferation and expression of platelet-derived growth
factor A chain gene in human vascular smooth muscle cells. FEBS
Lett 314:143
-148, 1992[Medline]
Albrightson CR, Nambi P, Zabko-Potapovich B, Dytko G, Groom T:
Effect of thrombin on proliferation, contraction and prostaglandin production
of rat glomerular mesangial cells in culture. J Pharmacol Exp
Ther 263:404
-412, 1992[Abstract/Free Full Text]
Shultz PJ, Knauss TC, Mene P, Abboud HE: Mitogenic signals for
thrombin in mesangial cells: Regulation of phospholipase C and PDGF genes.
Am J Physiol 257:F366
-F374, 1989[Abstract/Free Full Text]
He CJ, Rondeau E, Medcalf RL, Lacave R, Schleuning WD, Sraer JD:
Thrombin stimulates proliferation and decreases fibrinolytic activity of human
glomerular epithelial cells. J Cell Physiol146
: 131-140,1991[Medline]
Villamediana LM, Rondeau E, He CJ, Medcalf RL, Peraldi MN, Lacave
R, Delarue F, Sraer JD: Thrombin regulates components of the fibrinolytic
system in human mesangial cells. Kidney Int38
: 956-961,1990[Medline]
Tomooka S, Border WA, Marshall BC, Noble NA: Glomerular matrix
accumulation is linked to inhibition of the plasmin protease system.
Kidney Int 42:1462
-1469, 1992[Medline]
Wojta J, Gallicchio M, Zoellner H, Hufnagl P, Last K, Filonzi EL,
Binder BR, Hamilton JA, McGrath K: Thrombin stimulates expression of
tissue-type plasminogen activator and plasminogen activator inhibitor type 1
in cultured human vascular smooth muscle cells. Thromb
Haemostasis 70:469
-474, 1993[Medline]
Fujimoto N, Mouri N, Iwata K, Ohuchi E, Okada Y, Hayakawa T: A
one-step sandwich enzyme immunoassay for human matrix metalloproteinase-2 (72
kDa gelatinase/type IV collagenase) using monoclonal antibodies.
Clin Chem Acta 221:91
-103, 1993[Medline]
Zhang J, Fujimoto N, Iwata K, Sakai T, Okada Y, Hayakawa T: A
one-step sandwich enzyme immunoassay for human matrix metalloproteinase-1
(interstitial collagenase) using monoclonal antibodies. Clin Chem
Acta 219: 1-14,1993[Medline]
Lowry O, Rosenbrough NJ, Farr AL: Protein measurement with the
Folin phenol reagent. J Biol Chem193
: 265-275,1951[Free Full Text]
Feinberg AP, Vogelstein B: A technique for radiolabelling DNA
restriction endonuclease fragments to high specific activity. Anal
Biochem 132:6
-13, 1983[Medline]
Martin JM, Davies M, Thomas G, Lovett DH: Human mesangial cells
secrete a GBM-degrading neutral proteinase and a specific inhibitor.
Kidney Int 36:790
-801, 1989[Medline]
Kitamura M, Shirasawa T, Maruyama N: Gene transfer of
metalloproteinase transin induces aberrant behavior of cultured mesangial
cells. Kidney Int 45:1580
-1586, 1994[Medline]
Lovett DH, Johnson RJ, Marti HP, Martin J, Davies M, Couser WG:
Structural characterization of the mesangial cell type IV collagenase and
enhanced expression in a model of immune complex-mediated glomerulonephritis.
Am J Pathol 141:85
-98, 1992[Abstract]
Markwardt F: The development of hirudin as an antithrombotic drug.
Thromb Res 74:1
-23, 1994[Medline]
Vanholder RC, Camez AA, Veys NM, Soria J, Mirshahi M, Soria C,
Ringoir S: Recombinant hirudin: A specific thrombin inhibiting anticoagulant
for hemodialysis. Kidney Int45
: 1754-1759,1994[Medline]
Nelken NA, Soifer SJ, O'Keefe J, Vu TK, Charo IF, Coughlin SR:
Thrombin receptor expression in normal and atherosclerotic human arteries.
J Clin Invest 90:1614
-1621, 1992
Herbert JM, Lamarche I, Dol FTI: Induction of vascular smooth
muscle cell growth by selective activation of the thrombin receptor: Effect of
heparin. FEBS Lett 301:155
-158, 1992[Medline]
Vu TKJ, Hung DT, Wheaton VI, Coughlin SR: Molecular cloning of a
functional cloning receptor reveals a novel proteolytic mechanism of receptor
activation. Cell 64:1057
-1068, 1991[Medline]
Nahman NJ, Leonhart KL, Cosio FG, Hebert CL: Effects of high
glucose on cellular proliferation and fibronectin production by cultured human
mesangial cells. Kidney Int 41:396
-402, 1992[Medline]
Striker LM, Killen PD, Chi E, Striker GE: The composition of
glomerulosclerosis. Lab Invest51
: 181-192,1984
Ishimura E, Sterzel RB, Budde K, Kashgarian M: Formation of
extracellular matrix by cultured rat mesangial cells. Am J
Pathol 134:843
-855, 1989[Abstract]
Border WA, Okuda S, Languino LR, Ruoslahti E: Transforming growth
factor-beta regulates production of proteoglycans by mesangial cells.
Kidney Int 37:689
-695, 1990[Medline]
Border WA, Okuda S, Languino LR, Sporn MB, Ruoslahti E: Suppression
of experimental glomerulonephritis by antiserum against transforming growth
factor ß1. nature 346:371
-374, 1990[Medline]
Floege J, Johnson RJ, Gordon K, Iida H, Pritzl P, Yoshimura A,
Campbell C, Alpers CE, Couser WG: Increased synthesis of extracellular matrix
in mesangial proliferative nephritis. Kidney Int40
: 477-488,1991[Medline]
Sedor JR, Nakazato Y, Konieczkowski M: Interleukin-1 and the
mesangial cell. Kidney Int 41:595
-599, 1992[Medline]
Kim SB, Kang SA, Cho YJ, Park SK, Cheong HI, Lee JD, Hong CD, Park
JS: Effects of low density lipoprotein on type IV collagen production by
cultured rat mesangial cells. Nephron67
: 327-333,1994[Medline]
Couchman JR, Beavan LA, McCarthy KJ: Glomerular matrix: Synthesis,
turnover and role in mesangial expansion. Kidney Int45
: 328-335,1994[Medline]
Stetler S-W, Brown PD, Onisto M, Levy AT, Liotta LA: Tissue
inhibitor of metalloproteinases-2 (TIMP-2) mRNA expression in tumor cell lines
and human tumor tissues. J Biol Chem265
: 13933-13938,1990[Abstract/Free Full Text]
Wong AP, Cortez SL, Baricos WH: Role of plasmin and gelatinase in
extracellular matrix degradation by cultured rat mesangial cells.
Am J Physiol 263:F1112
-F1118, 1992[Abstract/Free Full Text]
Kitamura M, Maruyama N, Mitarai T, Nagasawa R, Yokoo T, Sakai O:
Heparin selectively inhibits gene expression of matrix metalloproteinase
transin in cultured mesangial cells. Biochem Biophys Res
Commun 203:1333
-1338, 1994[Medline]
Martin J, Knowlden J, Davies M, Williams JD: Identification and
independent regulation of human mesangial cell metalloproteinases.
Kidney Int 46:877
-885, 1994[Medline]
Marti HP, Lee L, Kashgarian M, Lovett DH: Transforming growth
factor-beta 1 stimulates glomerular mesangial cell synthesis of the 72-kd type
IV collagenase. Am J Pathol144
: 82-94,1994[Abstract]
Overall CM, Wrana JL, Sodek J: Independent regulation of
collagenase, 72-kDa progelatinase, and metalloendproteinase inhibitor
expression in human fibroblasts by transforming growth factor-ß.
J Biol Chem 264:1860
-1869, 1989[Abstract/Free Full Text]
Overall CM, Wrana JL, Sodek J: Transcriptional and
post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by
transforming growth factor-ß1 in human fibroblasts: Comparisons with
collagenase and tissue inhibitor of matrix metalloproteinase gene expression.
J Biol Chem 266:14064
-14071, 1991[Abstract/Free Full Text]
Border WA: Transforming growth factor-beta and the pathogenesis of
glomerular diseases. Curr Opin Nephrol Hypertens3
: 54-58,1994[Medline]
Floege J, Eng E, Young BA, Johnson RJ: Factors involved in the
regulation of mesangial cell proliferation in vitro and in vivo.
Kidney Int Suppl S47-S54,1993
Yamamoto T, Noble NA, Cohen AH, Nast CC, Hishida A, Gold LI, Border
WA: Expression of transforming growth factor-beta isoforms in human glomerular
diseases. Kidney Int 49:461
-469, 1996[Medline]
Yoshioka K, Takemura T, Murakami K, Okada M, Hino S, Miyamoto H,
Maki S: Transforming growth factor-beta protein and mRNA in glomeruli in
normal and diseased human kidneys. Lab Invest68
: 154-163,1993[Medline]
Sower LE, Froelich CJ, Carney DH, Fenton JW, Klimpel GR: Thrombin
induces IL-6 production in fibroblasts and epithelial cells: Evidence for the
involvement of the seven-transmembrane domain (STD) receptor for
alpha-thrombin. J Immunol 155:895
-901, 1995[Abstract]
Yamabe H, Osawa H, Inuma H, Kaizuka M, Tamura N, Tsunoda S, Baba Y,
Shirato K, Onodera K: Thrombin stimulates production of transforming growth
factor-ß by cultured human mesangial cells. Nephrol Dial
Transplant 12:438
-442, 1997[Abstract/Free Full Text]
Received for publication September 2, 1997.
Accepted for publication December 23, 1998.
Top
Abstract
Introduction
-treated human
skin fibroblasts (CCD-41SK; American Type Culture Collection [ATCC] no.
CRL1505) (28) and against a
synthetic peptide corresponding to the N-terminal domain (residues 17 to 35)
of proMMP-2. The sensitivity of this EIA was 0.24 ng/ml, and linearity was
obtained between 1.0 and 500 ng/ml. 
) of 5 U/ml thrombin. Thrombin
stimulated the production of type IV collagen (P < 0.0001) and
TIMP-1 (P < 0.005) in a time-dependent manner. Results are
expressed as ng per µg cell protein. Values are the mean ± SD for
four wells.
