© 1995 American Heart Association, Inc.
Articles |
Thrombin Induces the Preproendothelin-1 Gene in Endothelial Cells by a Protein Tyrosine Kinase–Linked Mechanism
From the Departments of Medicine (T.A.M., M.S.S., M.J.D.) and Physiology and Biophysics (M.J.D.), School of Medicine, Case Western Reserve University, Cleveland, Ohio, and the Division of Nephrology (M.J.D.), University Hospitals of Cleveland.
Correspondence to Dr Michael J. Dunn, Division of Nephrology, Department of Medicine, University Hospitals of Cleveland, 2074 Abington Rd, Room 8124, Lakeside Building, Cleveland, OH 44106.
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Abstract Thrombin stimulates synthesis and secretion of endothelin-1 (ET-1), a vasoactive peptide that triggers responses in the vascular endothelium and smooth muscle. We investigated the signal transduction pathways by which thrombin stimulates preproET-1 gene expression and ET-1 peptide secretion in macrovascular cells (human umbilical vein endothelial cells [HUVECs] and bovine pulmonary artery endothelial cells [BPAECs]) and microvascular cells (human microvascular endothelial cell line [HMEC-1]). Thrombin (4 U/mL) stimulated maximal induction of ET-1 peptide secretion and preproET-1 mRNA after 2 hours in HUVECs and BPAECs and after 1 hour in HMEC-1. A synthetic thrombin receptor activator peptide confirmed ligand-specific receptor actions to induce preproET-1 mRNA. Protein kinase C (PKC) activation by phorbol ester transiently induced preproET-1 mRNA but had no effect on ET-1 peptide synthesis. PKC inhibitors sangivamycin and calphostin C and PKC depletion failed to suppress thrombin-stimulated preproET-1 mRNA. Adenylate cyclase and cAMP-dependent protein kinase did not participate in thrombin-induced preproET-1 gene activation. Thrombin stimulated a rapid increase in phosphotyrosine-containing proteins, suggesting a role for tyrosine phosphorylation in thrombin signaling. These data demonstrate that thrombin induces the preproET-1 gene and ET-1 peptide synthesis by a PKC-independent PTK-dependent pathway in macrovascular and microvascular endothelial cells. Protein tyrosine kinase inhibitors herbimycin A and genistein blocked thrombin-stimulated preproET-1 mRNA and peptide secretion, whereas daidzein, which lacks inhibitory activity, did not suppress thrombin-induced ET-1.
Key Words: thrombin • endothelin • endothelial cells • protein tyrosine kinase • protein kinase C
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The vascular endothelium has diverse physiological activities, including the maintenance of a nonthrombogenic surface in blood vessels. However, in the presence of a variety of cytokines, hormones, physical forces, and vasoactive peptides, changes in cell shape, permeability, and focal disruption alter the endothelium, leading to vascular disorders. Thrombin, a procoagulant with widespread effects on hemostasis and in pathological vascular disorders such as ischemia and atherosclerosis,1 has been demonstrated to influence a variety of endothelial responses including the release of endothelin-1 (ET-1).2 3 Thrombin increases both ET-1 synthesis and preproET-1 mRNA levels; however, the exact mechanism by which thrombin stimulation leads to altered preproET-1 gene expression and ET-1 peptide secretion is currently not well understood.
Induction of the preproET-1 gene by thrombin in endothelial cells is complex and occurs by protein kinase C (PKC)–dependent and PKC-independent pathways. The preproET-1 gene can be transcriptionally regulated.2 4 5 In porcine aortic endothelial cells, thrombin induces preproET-1 mRNA via PKC,6 and immunoreactive ET-1 release in porcine aortic endothelial cells, after thrombin stimulation, can be inhibited by PKC inhibitors H-7 and staurosporine.7 Thrombin and phorbol esters stimulate immunoreactive ET-1 secretion in cultured bovine endothelial cells.8 In human venous endothelial cells, thrombin-stimulated ET-1 secretion is rapid and transient,9 and phorbol esters were used to link preproET-1 mRNA expression to PKC activation.10 In contrast, prolonged exposure to another agonist, shear stress, regulates preproET-1 gene transcription independent of PKC in bovine arterial endothelial cells.11
Recent evidence suggests that venous and arterial endothelial cells respond differently after thrombin stimulation; this difference may be due to two distinct thrombin receptor subtypes in venous and arterial endothelium.12 There have been no published results involving an identification or cloning of such a second class of thrombin receptors. Furthermore, controversy exists concerning the differences in ET-1 synthesis in macrovascular and microvascular endothelial cells, which are both known to produce immunoreactive ET-1.13 14 15 This may be especially important since the microvasculature is known to be more susceptible than the macrovasculature to vasoconstrictors and vasodilators and represents a major site for blood pressure regulation. Therefore, it remains to be clarified whether different stimuli act via distinct signaling pathways in different endothelial cell types to induce preproET-1 gene expression. Moreover, the PKC-independent pathways regulating preproET-1 gene expression have not been characterized.
The goal of the present study was to investigate endothelial cells of various origins for their ability to produce ET-1 and to analyze the intracellular signaling pathways involved. Special emphasis was placed on the contribution of protein tyrosine kinase (PTK)–dependent signal transduction pathways in preproET-1 gene induction after thrombin stimulation. We used cultured human umbilical vein endothelial cells (HUVECs), a human microvascular endothelial cell line (HMEC-1), and bovine pulmonary artery endothelial cells (BPAECs). We could demonstrate a novel signaling cascade for thrombin-stimulated preproET-1 gene expression and ET-1 peptide synthesis in these cells. Our data provide evidence that in all endothelial cells after thrombin stimulation a common receptor-mediated pathway that is protein kinase A (PKA) and PKC independent exerts its actions via phosphorylation and activation of PTKs.
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Materials and Methods |
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Endothelial Cell Cultures
HUVECs were obtained by primary culture of cells harvested from umbilical cords using the technique of Lewis et al.16 Briefly, umbilical cords (length, 15 to 20 cm) without clamp marks were rinsed with phosphate-buffered saline to remove blood, clamped at both ends, and filled with solution containing 0.06% trypsin and 0.2% EDTA (JRH Biosciences) for 15 minutes at 37°C. Cords were then massaged to loosen cells that were subsequently transferred to 75-cm2 tissue culture flasks coated with fibronectin (1 µg/cm2) (Collaborative Research) and grown in MCDB 107 medium (Sigma Chemical Co) supplemented with 20% fetal bovine serum (Hyclone Laboratories Inc), 30 µg/mL endothelial cell growth supplement (Collaborative Research), and 1% penicillin/streptomycin (JRH Biosciences). On average, two umbilical cords yielded the amount of cells sufficient for one flask. Cells were usually grown for 5 or 6 days before they were subcultured. Experiments were performed on confluent contact-inhibited cells that had been kept in MCDB 107 medium containing 1% fetal bovine serum for 24 hours to induce quiescence.
BPAECs were obtained from American Type Culture Collection (CCL-209) and grown in DMEM (JRH Biosciences) supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin for 4 or 5 days before they were subcultured. Experiments were performed on confluent contact-inhibited cells that had been kept in serum-free DMEM for 24 hours to induce quiescence.
Simian virus 40–transfected immortalized HMEC-117 was obtained from the Center for Disease Control, Atlanta, Ga, at passage 13 and grown in MCDB 131 medium (GIBCO BRL) supplemented with 15% fetal bovine serum, 10 ng/mL epidermal growth factor (Collaborative Research), 1 µg/mL hydrocortisone (Sigma), and 1% penicillin/streptomycin for 3 or 4 days before they were subcultured. Experiments were performed on confluent contact-inhibited cells that had been kept in serum-free DMEM for 24 hours to induce quiescence.
Experiments were performed in all cell lines after addition of various agonists and antagonists. Sangivamycin was supplied by the National Cancer Institute. Herbimycin A and 12-O-tetradecanoylphorbol 13-acetate (TPA) were from GIBCO BRL; forskolin, calphostin C, genistein, and daidzein, from Calbiochem; Rp cAMPS, from BioLog Life Science; TRAP42-55 (Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe), from Bachem; pertussis toxin and cholera toxin, from List Biological Laboratories; and dibutyryl cAMP (Bt2 cAMP) and MAS-7, from Biomol.
mRNA Extraction and Hybridization
Total cellular RNA was extracted from endothelial cells by the
acid guanidinium thiocyanate/phenol/chloroform method,18
denatured with 3% formaldehyde, and fractionated in a 1.2% agarose
gel before being transferred to a nitrocellulose membrane (Schleicher &
Schuell). Membranes were then hybridized with the 32P
(DuPont NEN)–labeled 1.2-kb human ET-1 cDNA insert19 and
the GAPDH cDNA (ATCC 57090) for verification of equal amounts of RNA
per well under hybridization conditions containing 50% formamide, 5x
SSPE, 0.5% SDS, 10% dextran sulfate, 1x Denhardt's solution, and
0.2 mg/mL salmon testes DNA at 42°C. After 24 hours, membranes were
washed twice with 2x SSPE/0.1% SDS for 15 minutes and once with 0.2x
SSPE/0.1% SDS before being exposed to Fuji RX medical x-ray film for 1
day at -72°C. The amount of mRNA expression was quantified by
densitometry using National Institutes of Health IMAGE
software on an Apple Macintosh computer equipped with a Microtech
scanner and corrected for GAPDH gene expression.
Radioimmunoassay of ET-1
Levels for ET-1 peptide secretion in endothelial cell culture
supernatants were quantified by radioimmunoassay in triplicate. Cell
supernatants were collected, lyophilized, and resuspended in
radioimmunoassay buffer consisting of 100 mmol/L
NaH2PO4, 0.05 mol/L NaCl, 0.1% bovine
serum albumin, 0.1% Triton X-100, and 0.01% NaN3. The
rabbit anti–ET-1 serum (Peninsula Laboratories) that was used showed
100% specificity for ET-1, 17% cross-reactivity to big ET-1, and 7%
reactivity to endothelin-2 and endothelin-3. Antiserum (100 µL) was
added to equal amounts of either 100 µL endothelin standards or
reconstituted cell supernatants and incubated for 24 hours before 100
µL [125I]ET-1 (DuPont NEN), at a final concentration of
3500 cpm, was added for 24 hours. Endothelin bound to the antibody was
immunoprecipitated with 100 µL anti-rabbit serum (Peninsula
Laboratories) before 100 µL normal rabbit serum (Peninsula
Laboratories) before being separated from unbound antibody by
centrifugation. Finally, the amount of radioactivity in the
immunoprecipitants was determined by gamma counting (Packard
Instruments). ET-1 standard curves revealed 50% displacement by 8 to
12 pg ET-1. Endothelin concentrations were calculated by computer-aided
processing of the counting data using a logit/log transformation of the
calibration curve and were corrected for protein concentration per dish
(BCA protein assay, Pierce).
Anti-Phosphotyrosine Immunoblot Assays
Phosphorylation of cellular proteins on tyrosine was
characterized exactly as described by Dubyak and
coworkers.20 Briefly, cells were solubilized in 150 µL
lysis buffer consisting of 20 mmol/L Tris-Cl, pH 8.0, 137 mmol/L NaCl,
2 mmol/L EDTA, 10% glycerol, 1% Triton X-100, 1 mmol/L
Na3VO4, 1 mmol/L phenylmethylsulfonyl
fluoride, and 1 mmol/L leupeptin, vortexed, and cleared of nuclei and
cell debris by a 4-minute centrifugation step in a microfuge. Total
protein extracts were size-fractionated in a 10% SDS-polyacrylamide
gel before being electroblotted to a nitrocellulose membrane. These
immunoblots were then incubated with affinity-purified horseradish
peroxidase–conjugated monoclonal anti-phosphotyrosine antibody (clone
PY20, ICN Biomedical) and washed twice with buffer containing 0.1 mol/L
Tris, 0.1 mol/L NaCl, and 0.1% Tween and once with buffer with the
addition of 0.2% SDS. Protein-antibody conjugates were visualized by a
chemiluminescence reagent (DuPont NEN) that uses horseradish peroxidase
to oxidize luminol before being exposed to Fuji RX medical x-ray film
for 5 minutes.
PKC Assay of Cell-Free Extracts
Endothelial cell monolayers were investigated for PKC by using a
modification of the method described by Heasley and
Johnson.21 Cells were stimulated with TPA alone or in
coincubation with the synthetic PKC pseudosubstrate PKC 19-36 (LC
Laboratories), lysed in ice-cold lysis buffer consisting of 100 mmol/L
NaCl, 25 mmol/L ß-glycerophosphate, pH 7.2, 10 mmol/L NaF, 20
µmol/L NaH2PO4, 0.1% Triton X-100, 10
mmol/L MgCl2, 1 mmol/L EGTA, 1 mmol/L
dithiothreitol, and 50 µmol/L phenylmethylsulfonyl fluoride, scraped,
and cleared of nuclei and cell debris by a 1-minute centrifugation step
at 10 000g. After a second centrifugation step at
100 000g for 20 minutes, the cytosolic and the particulate
fractions were assayed with a commercially available PKC assay system
(Amersham Life Science) based on PKC-catalyzed transfer of
-phosphate from [32P]ATP (DuPont NEN) to an epidermal
growth factor receptor binding domain as a PKC-specific phosphorylation
substrate. Labeled peptides were then separated from unlabeled peptides
on phosphocellulose filters, which were washed twice in 75 mmol/L
orthophosphoric acid for 10 minutes and once in 75 mmol/L
NaH2PO4 for 10 minutes, before peptide
phosphorylation was determined by scintillation counting of
32P and corrected for protein concentration.
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Thrombin Induces PreproET-1 mRNA and ET-1 Peptide Secretion in a Time-Dependent Manner via the Thrombin Receptor
Time-course experiments after thrombin (4 U/mL) stimulation were performed in HUVECs, HMEC-1, and BPAECs (Fig 1
). HUVECs
and BPAECs revealed peak preproET-1 mRNA levels after 2 hours of
incubation, whereas in HMEC-1 the maximum stimulatory effect was seen
after 1 hour. Longer incubation periods showed a gradual decline of the
message in all cell lines investigated. ET-1 peptide secretion
progressively increased over 120 minutes in all cell lines
(Table).
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To determine dose-dependent regulation of the preproET-1 gene after
agonist stimulation, we also performed experiments with various
thrombin concentrations (Fig 1
). Thrombin showed potent preproET-1 mRNA
induction in all endothelial cells after stimulation at concentrations
of 2 to 8 U/mL. Maximal induction occurred at a concentration of 4 U/mL
after 2 hours of stimulation in HUVECs, HMEC-1, and BPAECs. ET-1
peptide secretion was also maximal at this concentration in all cell
lines, with differences in ET-1 peptide secretion ranging from 2- to
10-fold increments among endothelial cells: HUVECs<HMEC-1<<BPAECs
(Table).
To confirm that thrombin induced preproET-1 mRNA by receptor
activation, a synthetic 14–amino acid thrombin receptor activator
peptide (TRAP42-55) that specifically activates the
thrombin receptor22 was added. Similar to thrombin,
TRAP42-55 induced preproET-1 mRNA in a time- and
dose-dependent manner, with maximum induction occurring after 1 hour of
incubation at 10-4 mol/L in HUVECs, in HMEC-1, and in
BPAECs (Fig 2). When corrected for GAPDH mRNA
expression, TRAP42-55 stimulated preproET-1 mRNA expression
similar to thrombin. However, TRAP42-55 failed to stimulate
ET-1 peptide secretion, suggesting that the small peptide does not
completely mimic thrombin-stimulated receptor activation (Table). Taken
together, these results indicate that thrombin receptor activation, in
diverse types of endothelial cells, stimulates expression of the
preproET-1 gene and ultimately increases secretion of ET-1 peptide.
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PreproET-1 mRNA Induction and ET-1 Peptide Secretion in Response to
Thrombin Involve a Pertussis Toxin–Sensitive G Protein
Thrombin couples to a G protein–linked receptor on endothelial
cells before preproET-1 gene induction and ET-1 peptide secretion. In
the cell lines investigated by us, 15 mmol/L NaF/5 µmol/L
AlCl3, a G protein activator, mimicked
thrombin-mediated induction of the preproET-1 mRNA (Fig 3). To further characterize the thrombin receptor–G
protein linkage, we inhibited Gi with pertussis toxin
before agonist stimulation and also stimulated Gs directly
with cholera toxin and assayed preproET-1 mRNA expression. In all three
cell lines (HUVEC, BPAEC, and HMEC-1), incubation with 1 µg/mL
pertussis toxin for 5 hours before agonist stimulation inhibited
preproET-1 mRNA expression (Fig 3). ET-1 peptide secretion in
pertussis toxin–treated cells showed no significant inhibition in
BPAECs, whereas inhibition was partial in HMEC-1 and complete in HUVECs
(Table). Additional experiments, measuring preproET-1 mRNA and ET-1
peptide secretion after cholera toxin (1µg/mL) for 5 hours, failed to
show Gs protein coupling to the thrombin receptor in all
cell lines investigated. These findings suggest that in
thrombin-stimulated endothelial cells, preproET-1 gene and ET-1 peptide
induction are mediated via a pertussis toxin–sensitive Gi
protein–coupled thrombin receptor mechanism.
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Thrombin-Stimulated PreproET-1 mRNA Induction Is Not Mediated
by PKA
Thrombin has previously been shown to stimulate adenylate cyclase
and PKA in endothelial cells.23 We evaluated the possible
involvement of PKA activation by thrombin in preproET-1 gene
regulation. We stimulated or inhibited the PKA signaling pathway and
measured preproET-1 gene expression and ET-1 peptide secretion to
determine its role in agonist stimulation of endothelial cells.
Direct stimulation of adenylate cyclase with forskolin (20 µmol/L)
for 2 hours alone failed to induce preproET-1 mRNA levels and ET-1
peptide synthesis (Fig 4 and Table). Bt2
cAMP (100 µmol/L) also did not stimulate preproET-1 mRNA. Rp cAMPS
(100 µmol/L), a synthetic cAMP analogue that competitively inhibits
PKA, failed to inhibit preproET-1 mRNA induction or ET-1 peptide
secretion by thrombin (Fig 4 and Table).
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Forskolin and Bt2 cAMP reduced the constitutive expression
of preproET-1 mRNA in all three types of endothelial cells (Fig 4), but
constitutive ET-1 peptide secretion (Table), thrombin-stimulated
preproET-1 mRNA, and ET-1 peptide stimulation (data not shown) were
unaffected. These findings suggest that the PKA signaling pathway is
not involved in thrombin-stimulated preproET-1 mRNA and ET-1 peptide
induction.
PreproET-1 Gene Induction in Response to Thrombin Is Independent of
PKC
The seven-pass transmembrane thrombin receptor has been
demonstrated to be linked to activation of PKC through the
phospholipase D and phospholipase C signaling pathway,24
and PKC activation and preproET-1 gene induction have been
linked.6 7 8 9 10 Activation of PKC by phorbol esters, PKC
depletion before agonist stimulation, and inhibition with PKC
antagonists were used to determine the role of PKC in thrombin-mediated
preproET-1 gene induction.
Short-term activation of PKC with TPA (0.1 µmol/L) showed a modest
increase of preproET-1 mRNA at 30 minutes, which declined rapidly to
below basal levels over 12 hours of incubation (Fig 5).
PreproET-1 mRNA, which was depressed from 1 hour to 12 hours, recovered
after 24 hours of TPA treatment, suggesting that acute activation and
chronic desensitization of PKC can regulate the preproET-1 gene.
Peptide synthesis did not differ significantly from control levels
either at 2 or at 24 hours after TPA exposure (Table). To confirm that
TPA stimulated PKC activity under the conditions of our experiments, we
assayed PKC substrate phosphorylation after TPA stimulation (Fig 6). We showed strong PKC activation after TPA by
demonstrating a shift in activity from the cytosolic to the particulate
fraction in endothelial cells. Peptide phosphorylation increased 2.7
times in HMEC-1 and 3.3 times in HUVECs. Specificity of kinase
activation was indicated by incubation of fractionated cell components
with a synthetic PKC pseudosubstrate peptide (PKC
19-36),25 which competes for PKC substrate phosphorylation
and abolishes PKC activity. PKC 19-36 reduced by 85% PKC peptide
phosphorylation of cytosolic material from HUVECs or HMEC-1. These
results provide evidence that TPA acutely activates PKC and preproET-1
mRNA but not ET-1 synthesis and secretion. Furthermore, PKC
desensitization after prolonged PKC activation significantly reduced
preproET-1 gene expression.
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We26 and others27 have previously
demonstrated that preincubation of cells with TPA markedly depletes
immunoreactive PKC. When endothelial cells were pretreated for 24 hours
with TPA to downregulate PKC, thrombin stimulation showed preproET-1
mRNA induction comparable to that found with thrombin alone (Fig 5).
ET-1 peptide secretion was also unaffected by PKC downregulation
(Table). Although these data provided further evidence for preproET-1
gene induction by thrombin being independent of PKC, additional
experiments were performed to confirm this evidence. Inhibition of PKC
with selective inhibitors sangivamycin (0.1 µmol/L) and calphostin C
(0.1 µmol/L) before thrombin stimulation failed to inhibit preproET-1
gene expression or ET-1 peptide secretion in HUVECs as well as in
HMEC-1 and BPAECs (Fig 7 and Table). Collectively, these
results suggest that PKC is not involved in preproET-1 gene induction
and subsequent peptide synthesis after thrombin stimulation in
macrovascular and microvascular endothelial cells.
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PTK-Based Mechanisms Contribute to PreproET-1 mRNA Induction by
Thrombin
Recent experiments suggest that G protein–coupled receptors
activate nonreceptor PTKs, which contribute to downstream events
involving transcription and gene expression in platelets, mesangial
cells, and fibroblasts.28 29 30 31 32 33 To determine whether PTK
activation in endothelial cells may lead to preproET-1 gene induction,
we selectively inhibited PTK by using herbimycin A (0.1
µmol/L)34 and genistein (6 µg/mL)35
before agonist stimulation in HUVECs, HMEC-1, and BPAECs (Fig 7).
Inhibition of PTK decreased preproET-1 mRNA expression in
thrombin-stimulated HUVECs, HMEC-1, and BPAECs. Thrombin-stimulated
ET-1 peptide synthesis was reduced by both PTK antagonists to control
levels in HUVECs and HMEC-1 and below control levels in BPAECs (Table).
These data suggest that PTK-dependent phosphorylation after thrombin
stimulation is an important second messenger in preproET-1 mRNA
induction in endothelial cells. The inactive analogue of genistein,
daidzein (6 µg/mL),35 which lacks inhibitory activity
for PTK, served as a negative control and revealed no change in
preproET-1 mRNA after a comparable incubation period in
thrombin-stimulated endothelial cells (Fig 7). ET-1 peptide secretion
after daidzein and thrombin reached similar levels compared with
thrombin stimulation alone in all endothelial cell types (Table). The
importance of a tyrosine kinase–dependent signal transduction pathway
for thrombin-mediated preproET-1 mRNA induction is supported in
additional experiments, which demonstrate that genistein and herbimycin
are not nonspecific inhibitors of preproET-1 mRNA induction. PreproET-1
mRNA induction by A23187 and transforming growth factor-ß is not
blunted by PTK inhibitors in HUVECs (Fig 8).
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To confirm that thrombin increased tyrosine phosphorylation in
endothelial cells, direct determination of phosphotyrosine proteins
after thrombin stimulation by immunoblot assay was performed by using
monoclonal anti-phosphotyrosine antibodies. These experiments
demonstrated overall changes of phosphotyrosine containing protein
fractions after 30 minutes of thrombin stimulation in human endothelial
cells (Fig 9).
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Taken together, these results provide strong evidence for a PTK-dependent signaling pathway for induction of the preproET-1 gene after thrombin stimulation in HUVECs, HMEC-1, and BPAECs.
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Discussion |
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In the present study, we were able to demonstrate that thrombin induces preproET-1 mRNA and ET-1 peptide levels via receptor-mediated activation of PTK in human and bovine endothelial cells. Although TPA-induced activation of PKC stimulated the preproET-1 gene, neither PKC nor PKA activation represents the underlying mechanism in thrombin-stimulated preproET-1 mRNA induction or ET-1 peptide synthesis.
Our results confirm previously reported data involving thrombin-stimulated preproET-1 mRNA synthesis and ET-1 peptide release in endothelial cells.6 7 8 9 10 36 The induction was time dependent, and thrombin stimulated preproET-1 mRNA to a similar extent in all endothelial cell lines investigated, with major differences occurring in ET-1 peptide synthesis. According to our data, arterial cells produce significantly more ET-1 peptide than venous endothelial cells, and microvascular cells outproduce macrovascular endothelial cells. Even though these data were highly reproducible, an element of uncertainty remains as to whether these endothelial cell lines accurately represent arterial, microvascular, and macrovascular endothelium in vivo. With the exception of HUVECs, the cells were originated by other investigators and either transformed or highly passaged, and their clonal nature may therefore not be authentic for the respective endothelium.
Thrombin and TRAP42-55 induced preproET-1 mRNA to comparable amounts, thus providing evidence for a receptor-mediated signaling event. However, TRAP42-55 induces preproET-1 mRNA earlier, which also declines earlier compared with thrombin. Similar data have been reported for p44 mitogen-activated protein kinase activation with a related peptide using CCL-39 cells.37 Although thrombin stimulated less peptide secretion in venous than in arterial cells, TRAP42-55 failed to induce ET-1 peptide secretion in endothelial cells. The reasons for these differences between thrombin and TRAP42-55 remain unclear. At present, thrombin receptor activation has not been shown to be reversible, and TRAP42-55 is a potent stimulus, like thrombin, for phospholipase C and phospholipase A2, suggesting generation of the same intracellular messengers. One explanation for the failure of TRAP42-55 to induce ET-1 peptide secretion in endothelial cells is either rapid association/dissociation of the agonist from the receptor as well as time-dependent termination of downstream signaling events. Alternatively, the short peptide TRAP42-55 may not activate all of the signals that thrombin does and therefore does not completely mimic thrombin-stimulated receptor activation. Another possibility may be the relative instability of TRAP42-55 and its rapid degradation in the presence of aminopeptidases.38 Other reports, showing data similar to ours, of the inability of TRAP42-55 to induce PDGF secretion despite strong gene activation speculate on the possible participation of a second, yet unknown, thrombin receptor entity that is not activated by the synthetic peptide.39 Despite the discrepancy between ET-1 gene expression and secretion, our data provide strong evidence for a thrombin receptor–mediated mechanism that leads to preproET-1 gene induction.
To our knowledge, this report is the first to demonstrate that thrombin-stimulated preproET-1 gene induction and ET-1 peptide synthesis in endothelial cells is mediated via PTK activation. This pathway has not yet been described for preproET-1 mRNA induction and ET-1 peptide synthesis. Thrombin-stimulated ET-1 peptide release in porcine and bovine aortic endothelial cells as well as in HUVECs has previously been suspected to be PKC dependent.6 7 8 9 10 36 One report addressed preproET-1 mRNA induction by thrombin and its dependence on kinases in endothelial cells,6 and evidence exists for PTK activation in response to thrombin in platelets, fibroblasts, and mesangial cells.28 29 30 32 33 These observations favor signaling pathways other than PKC activation, and one report has recently demonstrated that preproET-1 mRNA induction by shear stress stimulation occurs independent of PKC in endothelial cells.11
On the basis of our experiments, we conclude that preproET-1 gene
induction by thrombin is mediated via PTK activation. We were able to
verify the initial preproET-1 mRNA induction by acute exposure to TPA;
however, on the basis of our data, PKC is unlikely to be involved in
thrombin-stimulated preproET-1 gene activation and ET-1 synthesis at
time points 
60 minutes in endothelial cells. Our data, making use of
PKC downregulation by pretreatment with TPA for 24 hours and of
specific PKC inhibitors, link preproET-1 mRNA induction and ET-1
peptide synthesis in endothelial cells after thrombin stimulation to a
PKC-independent and PKA-independent signaling pathway.
According to our data, it is likely, as proposed for platelets, that the endothelial cell thrombin receptor interacts with an intracellular nonreceptor tyrosine kinase via G protein activation to transmit its signal into the nucleus.32 We have not yet attempted to determine which family of tyrosine kinases may be functionally coupled to the thrombin receptor, but recent evidence closely links thrombin to pp60c-src activation in platelets.30 Similar evidence has also been presented in rat and hamster fibroblasts; thrombin regulates p21ras through activation of a heterodimeric Gi protein, which possibly involves an intermediary, yet unidentified, tyrosine kinase.33 In addition to further elucidation of the characteristics of the signaling pathways involved, the physiological impact of these findings also needs clarification. The role of PTK is not as well characterized as that of other second messengers, but PTK is assumed to be important in transmembrane signaling, cell proliferation, and cell transformation.40 The observation that thrombin activates the preproET-1 gene through PTK in endothelial cells indicates that thrombin may contribute to the effects of PTK on pathological vascular disorders, ischemia, and atherosclerosis.
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Acknowledgments |
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This study was supported by a fellowship grant of the Deutsche Forschungsgemeinschaft (MA 1464/1-1 [Dr Marsen]) and by the National Institutes of Health (HL-22563 [Dr Dunn]). We gratefully acknowledge Carol de la Motte for her help in establishing HUVEC primary culture techniques as well as Husam Ghnaim for technical assistance with the radioimmunoassay. We also thank Dr Kenneth D. Bloch for providing the human ET-1 cDNA clone.
Received January 26, 1995; accepted March 10, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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