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*
Division of Nephrology, Department of Emergency and Transplantation,
University of Bari, Bari, Italy.
Institute of Human Anatomy, University of Bari, Bari, Italy.
Correspondence to Prof. Francesco P. Schena, Division of Nephrology, Department of Emergency and Transplantation, University of Bari, Polyclinic, Piazza Giulio Cesare, 11, 70124 Bari, Italy. Phone: 39-80-5592237; Fax: 39-80-5575710; E-mail: fp.schena{at}nephro.uniba.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of this study was to investigate the effect of FXa on mesangial cell proliferation and to evaluate the potential signaling pathways that are activated by this coagulation factor. Our data demonstrate that FXa induces DNA synthesis and PDGF gene expression in human mesangial cells. Interestingly, the FXa mitogenic action is PDGF independent and most likely involves its binding to a protease-activated receptor with consequent increase in cytosolic calcium ([Ca2+]i) and in tyrosine phosphorylation of cytoplasmic proteins.
Thus, we can propose FXa as a novel growth factor for human mesangial cells (HMC) and support the hypothesis of a pathogenic involvement of this coagulation factor in mesangioproliferative glomerulonephritides.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Isolation and Culture
Normal-appearing portions of human kidneys that were surgically removed for
renal carcinoma were used to culture mesangial cells from outgrowths of
collagenase-treated glomeruli. HMC were established and characterized as
previously reported (14).
Cells were grown until confluent in RPMI 1640 medium supplemented with 17%
heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, 2 mM sodium pyruvate, 1% (vol/vol)
nonessential amino acids, 5 µg/ml insulin, 5 µg/ml transferrin, and 5
ng/ml selenium. For passage, confluent cells were washed with PBS, removed
with 0.025% trypsin/0.5 mM ethylenediaminetetraacetate (EDTA) in PBS and
plated in RPMI. Experiments included in this study were performed on cells
between the 5th and 10th passages.
Culture Condition and Cell Growth
DNA synthesis was measured as the amount of
[methyl-3H]-thymidine incorporated into trichloroacetic acid
(TCA)-precipitable material as described previously
(14). Briefly, cells were
plated in 24-well dishes at a density of 4 x 104/well, grown
to confluence, and made quiescent by placing them in serum-free medium for 48
h. Before the serum-free medium was added, the cells were washed three times
with PBS to avoid any possible contamination with thrombin and/or prothrombin
present in the FBS. Then cells were incubated with FXa at the indicated
concentrations for 24 h at 37°C. In a separate set of experiments, cells
were preincubated with 10-7M PMA for 48 h, with genistein (25
µM) for 18 h, or with leupeptin (1 µg/ml) or neutralizing polyclonal
rabbit anti human PDGF AB antibody (50 µg/ml) for 30 min before adding FXa
for 24 h. At the end of the incubation period, cells were pulsed for 4 h with
1.0 µCi/ml 3H-thymidine. The medium then was removed, and the
cells were washed twice in ice-cold 5% TCA and incubated in 5% TCA for 5 min.
The cell layers were solubilized by adding 0.75 ml of 0.25 N NaOH in 0.1%
sodium dodecyl sulfate (SDS). Half milliliter aliquots were then neutralized
and counted in scintillation fluid using a ß counter.
RNA Isolation and Northern Blot Analysis
For each experiment, 2 x 106 cells were plated in
75-mm2 flasks and cultured as detailed above. After reaching
confluence, HMC were serum-starved for 48 h and then incubated for the
indicated time periods in serum-free insulin-free RPMI 1640 containing 10 nM
FXa. At the end of incubation, cells were lysed with 4 M guanidium
isothiocyanate containing 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and
0.1 mM 2-ß-mercaptoethanol. Total RNA was isolated by the single-step
method, using phenol and chloroform/isoamylalcohol
(15).
PDGF A chain gene expression was studied by Northern blotting as described previously (6). Briefly, electrophoresis of 20 µg of total RNA from each experimental condition was carried out in 1 % agarose gel with 2.2 M formaldehyde. The gel was transferred overnight to a nylon membrane (Schleicher & Schuell, Dassel, Germany). The membrane was stained with ethidium bromide to evaluate the 28-S and 18-S bands and then prehybridized at 42°C for 2 h in 50% Formamide, 0.5% SDS, 5x SSC, and 0.1 mg/ml salmon sperm DNA. The cDNA probe used was a 1.3-kb fragment encoding the human PDGF A chain isolated from pGEM1 plasmid with EcoRI. The probe was labeled by random priming using a commercially available kit (Amersham) and [32P]dCTP (specific activity, 3000 Ci/mmol). The 32P-labeled probe (106 cpm/ml) was added to 10 ml of prehybridization solution, and the blots were hybridized for 16 h at 42°C. The membranes were then washed once in 2x SSC, 0.1% SDS at room temperature for 5 min, once in the same buffer at 55°C for 30 min, and in 1x SSC, 0.1% SDS at 55°C for another 30 min. After drying, membranes were exposed to a Kodak X-OMAT film with intensifying screens at -70°C.
Reverse Transcription-PCR
HMC express extremely low levels of PDGF B chain-specific transcript, which
do not allow for conventional Northern blot analysis. Therefore, in
preliminary experiments, we tried to analyze the target gene expression using
a highly sensitive and specific RNase protection assay performed exactly as
described previously (16).
Unfortunately, this technical approach also failed to identify measurable
amounts of PDGF B chain mRNA in 100 µg of total RNA extracted from
unstimulated cells. Thus, we resolved to address this issue by adopting a
semiquantitative reverse transcription-PCR (RT-PCR), which allowed us to
compare the relative amounts of target gene transcripts in the different
experimental conditions selected.
One µg of total RNA from cultured HMC was used in an RT reaction. Twenty µ1 of the reaction mixture containing 1 µg of total RNA, PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl), 5 mM MgCl2, 1 mM dNTPs, 20 U of RNase inhibitor, 2.5 mM oligo (dT), and 100 U of Moloney murine leukemia virus reverse transcriptase were inactivate the enzyme activity and to denature RNA-cDNA hybrids. All samples were reverse transcribed in the same set of experiments, and the efficiency of the reaction was checked by glyceraldehyde phosphate dehydrogenase (GAPDH) amplification.
PCR was performed with two separate sets of oligonucleotide primers, specific for human PDGF B chain and GAPDH, respectively: PDGF B chain: 5'-ATG CTG AGT GAC CAC TCG-3' upstream, 5'-CCT GAA TTT CCG GTG CTT GCC-3' downstream; GAPDH: 5'-TGG TAT CGT GGA AGG ACT CAT GAC-3' upstream, 5'-ATG CCA GTG AGC TTC CCG TTC AGC-3' downstream.
PDGF B chain or GAPDH cDNA amplification was performed in two separate sets of reactions at a final concentration of 1x PCR buffer, 1.5 mM MgCl2, 200 µM dNTPs, 0.15 µM PDGF B primers or 0.15 µM GAPDH primers, and 1.25 U of AmpliTaq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT), in a total volume of 50 µl. The amplification profile involved denaturation at 95°C for 1 min, primer annealing at 61°C for PDGF B or at 55°C for GAPDH for 1 min, and extension at 72°C for 1 min. PCR products were electrophoresed in 1.6% agarose gel in Tris borate/EDTA buffer, loading 10 µl of either PDGF B or GAPDH PCR products for each sample. The expected size of the amplified fragments was 588 bp and 450 bp for PDGF B chain and GAPDH, respectively.
In a separate set of experiments, total RNA from growing HMC was reverse transcribed as described and amplified using the following sets of primers specific for human EPR-1 and ß-actin: EPR-1: 5'-TTACGCCAGACTTCAGCCTG-3' upstream, 5'-TGGGTAACAGTGGCTGCTTC-3' downstream; ß-actin: 5'-GGCATCGTGATGGACTCCG-3' upstream, 5'-GCTGGAAGGTGGACAGCGA-3' downstream. The expected size of the fragments was 295 bp and 746 bp for the two EPR-1 transcripts and 700 bp for ß-actin.
Western Blot Analysis
HMC were grown in 60-mm2 Petri dishes to confluence in RPMI
containing 17% FBS. The cell monolayer was incubated 48 h in serum-free medium
and then exposed to 10 nM FXa for the indicated time periods. At the end of
the treatment, the cell monolayer was rinsed rapidly twice with ice-cold PBS
and lysed in 100 µl of RIPA buffer (1 mM phenylmethylsulphonyl fluoride, 5
mM EDTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 8 µg/ml
leupeptin, 1.5% nonidet P-40, 20 mM tris-HCl [pH 7.4]). The lysates were set
on ice for 30 min and centrifuged at 12,000 x g at 4°C for
5 min, and the supernatants were collected and stored at -80°C until used.
Aliquots containing 7.5 µg of proteins from each lysate were subjected to
SDS-polyacrylamide gel electrophoresis on 7.5% gels under reducing conditions
and then electrotransferred onto nitrocellulose membranes (Hybond C;
Amersham). After blocking nonspecific binding through incubation with 2%
bovine serum albumin and 0.1% Tween-20 in PBS (TBS) overnight at room
temperature, the membranes were incubated with monoclonal antiphosphotyrosine
antibody Py20 at room temperature for 4 h. The membranes were washed twice in
TBS and then incubated with horseradish-peroxidase-conjugated sheep anti-mouse
antibody at 1:1500 dilution in 0.1% tween-20 in PBS for 2 h at room
temperature. The membranes were washed three times at room temperature in TBS
and then once with 0.1% SDS in PBS. The ECL enhanced chemiluminescence system
was used for detection.
Immunoprecipitation and Anti-Phospho JNK Immunoblotting
Confluent HMC in 60-mm2 culture dishes were placed in serumfree
medium for 48 h. FXa was then added for the indicated time periods. Cells were
washed twice with ice-cold PBS and lysed with RIPA buffer for 30 min at
4°C. The cell lysate was centrifuged at 10,000 x g for 30
min at 4°C. A total of 100 µg of protein from the supernatant first was
incubated with antiphosphotyrosine antibodies for 2 h on a rocking platform at
4°C and then with agarose-linked protein A for 2 h at 4°C. The
immunobeads were washed twice with RIPA buffer and twice with 0.5 mM LiCl, 0.1
mM Tris-HCl (pH 7.5), and 1 mM sodium orthovanadate. The beads were then
resuspended in sample buffer and boiled. The immunoprecipitated proteins were
separated by electrophoresis on a 10% polyacrylamide gel and transferred onto
a nitrocellulose membrane. The membrane was blocked as described and incubated
with mouse anti-phospho JNK antibody (1:500) for 4 h at room temperature,
washed, and incubated with horseradish peroxidase-conjugated rabbit anti-mouse
IgG antibody (1:1500). The ECL enhanced chemiluminescence system was used for
detection.
Measurements of [Ca2+]i
[Ca2+]i was measured at 37°C by dual-wavelength
fluorescence microscopy in groups of 7 to 10 cells loaded with the
Ca2+ -sensitive indicator fura 2, as described previously
(17). Briefly, cells seeded at
a density of 2 x 104/24 mm diameter round glass coverslips
were loaded with 3 to 10 µm of fura 2 acetoxymethylester in serum-free
medium at 37°C and 5% CO2 for 60 min. Coverslips were rinsed
twice and then incubated subsequently with FXa and thrombin at 37°C in
Krebs-Ringer-HEPES buffer (125 mm NaCl, 5 mm KCl, 1.2 mm
KH2PO4, 1.2 mm MgSO4, 2 mm CaCl2,
265 mm HEPES, 6 mm glucose). Cells were not washed between the two different
stimuli. [Ca2+]i-dependent fluorescence was measured
with a microfluorometer (Cleveland Bioinstrumentation, Cleveland, OH)
connected to a Zeiss IM35 inverted microscope equipped with a Nikon CF X40
fluor objective. Recordings were performed at 340- and 380-nm excitation
wavelengths (bandwidth, 0.5 nm). Emission was collected by a photomultiplier
carrying a 505-nm cutoff filter. Emission from 340 and 380 nm and real-time
340- to 380-nm ratios were recorded by a specific softwere (Labview, National
Instruments Corporation, Austin, TX). In each experiment, the maximal
fluorescence ratio was determined by addition of Ca2+ ionophore
ionomycin, and the minimal fluorescence ratio was determined by addition of
7.5 mM ethyleneglycol-bis(ß-aminoethyl
ether)-N,N'-tetraacetic acid and 60 mM Tris (hydroxymethyl)
aminomethane HCl (pH 10.5). Fura 2 dissociation constant was assumed to be 224
nM. [Ca2+]i was calculated using previously described
formulas (18).
Statistical Analyses
Data are presented as mean ± SD. Data were compared using ANOVA or
two-tailed unpaired t test, as appropriate. P < 0.05 was
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects on [Ca2+]i Mobilization
Activation of the four known PAR results in the inducation of phospholipase
C (PLC) activity with the subsequent increase in intracellular calcium
concentration
(8,9,10,11).
To investigate the intracellular mitogenic signaling pathways elicited by FXa,
we studied the activation of PLC indirectly by measuring the cytosolic calcium
levels in groups of 7 to 10 human mesangial cells stimulated with FXa (10 nM).
Increases in [Ca2+]i rapidly occurred, peaked within a
few minutes, and declined slowly to reach a plateau at a slightly elevated
steady-state level, which was maintained for several minutes
(Figure 2A). After the early
response to FXa was exhausted, the same cells were stimulated with thrombin
(10 U/ml) to evaluate the activation and consequent downregulation of thrombin
receptors by FXa in HMC. The FXa-stimulated cells were still responsive to
thrombin, which induced an increase in [Ca2+]i following
a kinetic similar to that previously described
(4). The response mirrored the
pattern described for FXa stimulation but with a higher peak and a more
sustained plateau. To confirm the ability of FXa to elicit a
[Ca2+]i spike despite the downregulation of thrombin
receptors, we repeated the same experiments exposing the cells first to
thrombin and then to FXa. The downregulation of thrombin receptors did not
influence the cells' responsiveness to FXa
(Figure 2B). Conversely, both
FXa and thrombin completely downregulated their own response in HMC
(Figure 2, C and D).
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Effect of FXa on Intracellular Tyrosine Kinases
Tyrosine phosphorylation is a key event in the intracellular signaling of
different mitogens (19).
Recently, non-tyrosine kinase (non-TK) receptor agonists, including thrombin,
were shown to induce tyrosine phosphorylation of several cellular proteins
(19). Therefore, the ability
of FXa to induce the activation of intracellular TK was evaluated by
immunoblotting using a specific monoclonal antiphosphotyrosine antibody. FXa
(10 nM) caused a time-dependent increase in tyrosine phosphorylation of a
panel of cellular proteins. The apparent molecular weight of the most
prominent tyrosine-phosphorylated proteins was 140, 100, 80, 60, and 44 kD
(Figure 3).
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FXa has been shown to activate p44 mitogen-activated protein kinase in different cell types. In an attempt to identify one of the TK substrates phosphorylated in response to FXa, we blotted the antiphosphotyrosine immunoprecipitate with a specific anti-phospho JNK antibody, recognizing the activated JNK, another member of the MAP kinase family. As shown in Figure 4, FXa caused a rapid and significant increased in phosphorylated JNK in the antiphosphotyrosine immunoprecipitate, strongly suggesting a TK-dependent activation of this signaling enzyme induced by FXa.
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Effect of FXa on PDGF mRNA Expression
PDGF is one of the most powerful growth factors for HMC, and most of the
other mitogens, including thrombin, induce the expression of its two subunits
(4,
20). We sought to determine
whether FXa was able to stimulate PDGF A and B chain gene expression in
quiescent HMC. Figure 5
demonstrates the time course of changes that occurred in PDGF A chain mRNA
expression, studied by Northern blotting, in HMC exposed to FXa (10 nM). When
compared with control cells, FXa-stimulated HMC exhibited a marked increase of
PDGF A chain mRNA starting at 3 h, with a peak at 6 h, returning to near basal
levels by 12 h and increasing again at 24 h without reaching the maximum
expression found at 6 h. Then, we determined the effect of FXa on PDGF B chain
mRNA. As shown in Figure 6, FXa
also induced PDGF B chain mRNA expression with a peak at 6 h and a subsequent
reduction to basal level after 12 h.
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Regulation of FXa-Induced Mitogenesis
To determine whether the enzymatic activity of FXa was necessary to elicit
the mitogenic effect observed, we evaluated the effect of a specific
serine-protease inhibitor, leupeptin, on DNA synthesis stimulated by FXa.
Leupeptin at the concentration of 1 µg/ml completely abolished the increase
in thymidine uptake induced by FXa (Figure
7) without affecting the basal level (data not shown). To
investigate whether FXa mitogenic effect was dependent on thrombin release, we
used a specific thrombin inhibitor, hirudin. This antithrombin molecule at the
concentration of 20 UI/ml did not affect FXa-induced DNA synthesis
(Figure 7).
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Because FXa induced both PDGF A and B chain mRNA expression in human mesangial cells, we investigated whether its proliferative effect was dependent on the autocrine effect of PDGF. For this purpose, we studied FXa-induced DNA synthesis in HMC in the presence of a polyclonal neutralizing anti-human PDGF antibody (50 µg/ml) coincubated with FXa (10 nM). No significant reduction was observed in FXa-triggered thymidine incorporation (Figure 6), whereas the mitogenic response to PDGF AB (10 ng/ml) was completely abolished (control, 555 ± 50; PDGF AB, 2400 ± 320; PDGF AB + neutralizing antibody, 625 ± 115).
To understand the role of the increased calcium mobilization and protein tyrosine phosphorylation in FXa-induced mitogenesis in HMC, we examined the effect of Ca2+-dependent protein kinase C (PKC) downregulation and TK inhibition on DNA synthesis stimulated by FXa. The PKC downregulation, obtained as previously demonstrated (21), by a prolonged preincubation with PMA, significantly, although not completely, reduced thymidine uptake stimulated by FXa (Figure 7). Finally, genistein, an isoflavone compound that specifically inhibits TK, only partially reduced FXa-induced DNA synthesis (Figure 7), whereas a more pronounced and significant inhibition was observed with herbimycin A, a more powerful TK inhibitor.
EPR-1 Expression in HMC
Several reports support the hypothesis that the transmembrane protein EPR-1
is the potential FXa receptor. Interestingly, EPR-1 gene transcript undergoes
an alternative splicing with the production of two mature mRNA, the longer one
encoding a nuclear protein and the shorter the potential FXa receptor. We then
investigated by RT-PCR whether HMC express EPR-1. HepG2, a hepatocarcinoma
cell line, express both EPR-1 isoforms, whereas in human mesangial cells we
observed only the expression, at very low levels, of the transcript encoding
the nuclear protein (Figure
8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The availability of these factors in the glomerular microenvironment is indicated by the presence of prominent fibrin deposits during several forms of glomerular diseases (3). Despite this observation, the possible role of the coagulation proteases in the activation of glomerular cells is still largely unknown. Mesangial cells are specialized glomerular pericytes that are intimately involved in the hemodynamic regulation of the glomerular microvascular bed. Proliferation of these cells is a histopathologic hallmark of a variety of experimental and human glomerular diseases, and it has been implicated in the progression of glomerular injury toward sclerosis. The data reported in this study demonstrate, for the first time, that FXa activates HMC, inducing mitogenesis and PDGF A and B chain gene expression. Interestingly, FXa exerts its proliferative effect at doses even lower than its physiologic plasma concentrations (15 to 75 nM), with a significant increase in thymidine uptake starting at 2.5 nM. Therefore, we can hypothesize that in vivo, the small amounts of FXa locally produced by activating the coagulation system during glomerular damage could be sufficient to induce mitogenesis in HMC.
PDGF is the most powerful mitogen for HMC (26), and its synthesis always has been associated in vitro as well as in vivo with the proliferation of this cell line (20). Indeed, an increased expression of PDGF B chain and PDGF ß receptor has been demonstrated in experimental and human mesangioproliferative glomerulonephritis (27,28,29,30). Although earlier data (12), confirmed by recent findings (24), showed that FXa stimulates the release of PDGF from endothelial and vascular SMC, the effect of this serine-protease on PDGF A and B chain gene expression has not yet been investigated. Ko et al. (24) suggested that all of the effects induced by FXa in rat SMC were due to the release of PDGF, because neutralizing anti-PDGF antibodies were able to abolish any cell response to this factor. We excluded that FXa-induced HMC proliferation was caused by an autocrine effect of PDGF de novo synthesized or released by intracellular storage. Although FXa-induced thymidine uptake in the presence of a polyclonal anti-human PDGF antibody was not reduced significantly, we could not exclude an intracytoplasmic autocrine loop. However, our data on FXa-induced tyrosine phosphorylation do not reveal the presence of a phosphorylated protein of 170 kD corresponding to PDGF receptors. In addition, we found that anti-PDGF antibody does not inhibit FXa-induced tyrosine phosphorylation, further supporting the hypothesis that PDGF molecules eventually available are not responsible for cellular response mediated by tyrosine phosphorylation. Conversely, we cannot rule out definitively a role for other growth factors that potentially are secreted in response to FXa, including bFGF. However, the signaling events observed within a few minutes or seconds after the addition of FXa to the culture medium strongly suggest that this protease may stand on its own as a mitogenic factor.
Once a direct FXa mitogenic effect on HMC was demonstrated, the next question to answer concerned the receptor used and the intracellular signaling pathways elicited by this serine protease. Recently, Altieri (31) cloned in a leukemia cell line a cell surface receptor, called EPR-1, that specifically binds FXa. This receptor presents a single transmembrane domain and a short cytoplasmic tail with different potential serine-phosphorylation sites. Herbert et al. (32) demonstrated in human aortic SMC that a blocking anti—EPR-1 antibody is able to inhibit FXa-induced rabbit SMC growth. However, our cells do not express EPR-1 as demonstrated by RT-PCR. This finding, together with the observation that a nonspecific protease inhibitor completely blocks the mitogenic effect of FXa, suggests the potential involvement of a protease-activated receptor. There is now a growing interest in PAR that, for their structure and functions, belong to the G protein-coupled receptor superfamily. Besides the first thrombin receptor, in the last few years, three more PAR have been cloned and characterized (9,10,11). Two of these transmembrane proteins can interact specifically with thrombin, although they are not expressed at the renal level. These receptors, activated through limited proteolysis of the amino-terminal sequence, are able to induce an increase in [Ca2+]i (4,5,6), the activation of PKC (21), the modulation of gene expression, and the synthesis of DNA (22). We investigated whether the proteolytic activity of FXa was necessary to elicit the mitogenic effect on HMC. The complete abolition of FXa-induced DNA synthesis by leupeptin, a specific serine protease inhibitor, indicates that this enzyme requires its enzymatic activity to trigger cellular response. This observation suggests the interaction with a membrane receptor whose activation would occur by a proteolytic cleavage. Activation of all four known PAR results in the induction of PLC activity with subsequent intracellular calcium mobilization (8,9,10,11). We demonstrated for the first time that FXa stimulates an increase in [Ca2+]i at physiologic concentrations in HMC. The kinetics observed mirrored the one described previously for thrombin (4). FXa induced, within 1 min, a peak followed by a plateau at a slightly elevated steady-state level. This suggests an early release from intracellular stores and a later influx of extracellular calcium. Interestingly, we observed that HMC, soon after stimulation with FXa at doses that maximally induced DNA synthesis, maintain their responsiveness to thrombin. On the basis of this observation, it is conceivable that FXa does not occupy the same thrombin receptor(s), which therefore remain available to interact with this serine protease and to induce a spike of [Ca2+]i. As expected, stimulation of HMC first with thrombin and then with FXa showed the same pattern of [Ca2+]i release observed using the opposite experimental conditions. This finding strongly supports the involvement of two different classes of receptors in the cell interaction with these serine proteases. Indeed, Vu et al. (8) demonstrated that thrombin receptor, PAR-1, PAR-3, and PAR-4 are highly specific for thrombin and are poorly activated by other proteases. Nystedt et al. (9) found that PAR-2 is specifically activated by trypsin. Thus, another unknown PAR could be involved in the FXa-induced cellular effects.
The activation of PLC in FXa-stimulated HMC demonstrated indirectly by the
[Ca2+]i mobilization leads to PKC activation. To
evaluate the involvement of Ca2+-dependent PKC in HMC proliferation
induced by FXa, we downregulated it by a prolonged preincubation with PMA
(21), finding a significant
but not complete reduction of mitogenesis. This observation suggests an
involvement of Ca2+-dependent PKC but does not exclude that other
PKC isozymes, not affected by PMA preincubation, such as PKC
(21), could be responsible of
the residual mitogen effect. Interestingly, downregulation of PKC in the
presence of PMA did not result in any significant effect on thrombin-induced
DNA synthesis (21), suggesting
a divergence in the intracellular pathways elicited by these two serine
proteases.
Because the activation of the PLC-PKC axis could not explain fully the mitogenic effect of FXa, we sought to investigate possible influence of FXa-cell interaction on tyrosine kinase activation, a key intracellular pathway leading to mitogenesis. Increasing evidence demonstrates that a variety of agonists, including thrombin, in addition to activating the G-protein cascade, require tyrosine phosphorylation to induce mitogenesis (33,34,35). Our data demonstrate that FXa causes a time-dependent increase in tyrosine phosphorylation of a panel of HMC proteins, and this intracellular event is necessary for the FXa mitogenic effect. Indeed, the TK inhibitor genistein reduced significantly, although not completely, FXa-induced DNA synthesis. The possible interaction between PLC-PKC and TK signaling pathways remains unclear. In mesangial cells, thrombin-induced intracellular calcium mobilization is unaffected by TK inhibition (36). However, it has been reported that an increase in intracellular calcium, caused by angiotensin II or by a calcium ionophore, stimulates the tyrosine phosphorylation of several cellular proteins (37). Because FXa-induced tyrosine phosphorylation does not occur rapidly but has slow kinetics, it is conceivable that a cytoplasmic TK could be activated by an increase in [Ca2+]i concentration through a calcium-binding regulatory protein.
Although the precise identity of most tyrosine phosphorylated proteins remains to be determined, the 44- to 46-kD protein most likely may represent one of the MAP kinase isoforms. Indeed, extracellular signal-regulated kinase-1 activation by FXa was demonstrated in rat vascular SMC mitogenesis (24). Interestingly, FXa in HMC induces the phosphorylation of another kinase belonging to the MAP kinase superfamily, JNK. JNK is a downstream target of several cytoplasmic TK and can activate, through jun phosphorylation, the transcription factor activator protein-1 that, in turn, can modulate the expression of several genes (38).
In conclusion, we demonstrated for the first time that FXa is a powerful mitogenic stimulus for HMC and induces PDGF A and B chain gene expression. Its proliferative effect, requiring its proteolytic activity, may be mediated by a protease-activated receptor through the activation of the PLC-PKC and TK signaling pathways. Our findings suggest that FXa, through the biologic effects described here, could be responsible for the activation of mesangial cells during glomerular injury.
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Acknowledgments |
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References |
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regulates thrombin-induced PDGF-B chain gene
expression in mesangial cells. FEBS Lett373
: 146-150,1995[Medline]
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