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(Circulation Research. 1995;76:987-995.)
© 1995 American Heart Association, Inc.

Articles

Thrombin Induces the Preproendothelin-1 Gene in Endothelial Cells by a Protein Tyrosine Kinase–Linked Mechanism

Tobias A. Marsen, Michael S. Simonson, Michael J. Dunn

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.

	Abstract

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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

	Introduction

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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.

	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; TRAP^42-55 (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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
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).

View larger version (37K): [in this window] [in a new window]   Figure 1. Top, Representative Northern blot analysis of total cellular RNA hybridized with endothelin-1 (ET-1) cDNA and GAPDH cDNA in human umbilical vein endothelial cells (HUVECs) that had been exposed to thrombin in a time- and dose-dependent manner as indicated. Prepro- ET-1 mRNA showed maximal induction at 120 minutes of incubation with 4 U/mL thrombin. Contr indicates unstimulated control cells; conc, concentration. Bottom, Graphs showing results of densitometric analysis of Northern blots hybridized with ET-1 cDNA and normalized to corresponding GAPDH signal expression in HUVECs, a human microvascular endothelial cell line (HMEC-1), and bovine pulmonary artery endothelial cells (BPAECs) that had been exposed to thrombin in a time- and dose-dependent manner. Data shown represent three to eight experiments each. Bars for SEM were not included in the figure for clarity but did not exceed 0.9-fold in HUVECs, 1.8-fold in HMEC-1, and 0.7-fold in BPAECs during time-course and dose-response experiments.

View this table: [in this window] [in a new window]   Table 1. Immunoreactive Endothelin-1 Peptide Secretion in Endothelial Cell Supernatants

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 (TRAP^42-55) that specifically activates the thrombin receptor²² was added. Similar to thrombin, TRAP^42-55 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, TRAP^42-55 stimulated preproET-1 mRNA expression similar to thrombin. However, TRAP^42-55 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.

View larger version (38K): [in this window] [in a new window]   Figure 2. Top, Representative Northern blot analysis of total cellular RNA hybridized with endothelin-1 (ET-1) cDNA and GAPDH cDNA in human umbilical vein endothelial cells (HUVECs) that had been exposed to TRAP42-55 (Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) in a time- and dose-dependent manner as indicated. For comparison, lanes are shown for unstimulated control cells (Contr) as well as for thrombin (Thr, 4 U/mL for 120 minutes). Conc indicates concentration. Note that preproET-1 mRNA induction is equally strong for TRAP42-55 and Thr-treated cells and that in TRAP42-55-stimulated cells maximal gene induction occurs after 60 minutes. Bottom, Graphs showing results of densitometric analysis of Northern blots hybridized with ET-1 cDNA and normalized to corresponding GAPDH signal expression in HUVECs, a human microvascular endothelial cell line (HMEC-1), and bovine pulmonary artery endothelial cells (BPAECs) that had been exposed to TRAP42-55 in a time- and dose-dependent manner. Data shown represent three experiments each. Bars for SEM were not included in the figure for clarity but did not exceed 0.4-fold in HUVECs, 0.6-fold in HMEC-1, and 1.7-fold in BPAECs during time-course and dose-response experiments.

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 G_i with pertussis toxin before agonist stimulation and also stimulated G_s 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 G_s 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 G_i protein–coupled thrombin receptor mechanism.

View larger version (46K): [in this window] [in a new window]   Figure 3. Representative Northern blot analysis of total cellular RNA hybridized with endothelin-1 (ET-1) cDNA and GAPDH cDNA in human umbilical vein endothelial cells (HUVECs) exposed to cholera toxin (1 µg/mL for 5 hours), to cholera toxin (1 µg/mL for 5 hours) before thrombin (4 U/mL for 20 minutes), to pertussis toxin (1 µg/mL for 5 hours) before thrombin (4 U/mL for 120 minutes), and to NaF/AlCl3 (15 mmol/L for 120 minutes). For comparison, lanes are shown for unstimulated control cells as well as for cells exposed to thrombin (4 U/mL for 120 minutes). Densitometric analysis data for preproET-1 mRNA, normalized to corresponding GAPDH signal expression, are shown underneath each lane. Results for preproET-1 mRNA expression in a human microvascular endothelial cell line (HMEC-1) and bovine pulmonary artery endothelial cells (BPAECs) were not significantly different from results in HUVECs (data not shown).

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).

View larger version (35K): [in this window] [in a new window]   Figure 4. Representative Northern blot analysis of total cellular RNA hybridized with endothelin-1 (ET-1) cDNA and GAPDH cDNA in endothelial cells exposed to forskolin (20 µmol/L for 120 minutes), dibutyryl cAMP (Bt2 cAMP, 100 µmol/L for 120 minutes), and Rp cAMPS (100 µmol/L for 30 minutes) before thrombin (4 U/mL for 120 minutes). For comparison, lanes are shown for unstimulated control cells as well as for cells exposed to thrombin (4 U/mL for 120 minutes). Densitometric analysis data for preproET-1 mRNA, normalized to corresponding GAPDH signal expression, are shown underneath each lane.

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.

View larger version (49K): [in this window] [in a new window]   Figure 5. Representative Northern blot analysis of total cellular RNA hybridized with endothelin-1 (ET-1) cDNA and GAPDH cDNA in human umbilical vein endothelial cells (HUVECs) exposed to 0.1 µmol/L 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 hours and after 24 hours of incubation before exposure to thrombin (Thr, 4 U/mL for 120 minutes). For comparison, lanes are shown also for unstimulated control cells (Contr) as well as for cells exposed to Thr (4 U/mL for 120 minutes). Densitometric analysis data for preproET-1 mRNA, normalized to corresponding GAPDH signal expression, are shown underneath each lane. Results for preproET-1 mRNA expression in a human microvascular endothelial cell line and bovine pulmonary artery endothelial cells were not significantly different from results in HUVECs (data not shown). Note the initial preproET-1 mRNA increase at 30 minutes, followed by a rapid decline to undetectable amounts. PreproET-1 mRNA recovers after 24 hours and in this PKC-depleted state can be stimulated by Thr.

View larger version (18K): [in this window] [in a new window]   Figure 6. Graph showing time-course analysis of protein kinase C (PKC) peptide phosphorylation and translocation of activated peptide from the cytosol to the membrane, as well as inhibition of the cytosolic fraction with the truncated PKC pseudosubstrate PKC (19-36)22 after 0.1 µmol/L 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulation in a human microvascular cell line. Data represent five experiments each and show 2.7-fold increase in response to TPA (3.3-fold increase in human umbilical vein endothelial cells; data not shown).

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.

View larger version (31K): [in this window] [in a new window]   Figure 7. Representative Northern blot analysis of total cellular RNA hybridized with endothelin-1 (ET-1) cDNA and GAPDH cDNA in endothelial cells exposed to herbimycin A (1 µmol/L for 18 hours) before thrombin (4 U/mL for 120 minutes), genistein (6 µg/mL for 30 minutes) before thrombin (4 U/mL for 120 minutes), daidzein (6 µg/mL for 30 minutes) before thrombin (4 U/mL for 20 minutes), and sangivamycin (0.1 µmol/L for 30 minutes) before thrombin (4 U/mL for 120 minutes). HUVEC indicates human umbilical vein endothelial cells; HMEC-1, a human microvascular endothelial cell line; and BPAEC, bovine pulmonary artery endothelial cells. For comparison, lanes are shown for unstimulated control cells as well as for cells exposed to thrombin (4 U/mL for 120 minutes). Densitometric analysis data for preproET-1 mRNA, normalized to corresponding GAPDH signal expression, are shown underneath each lane. Data for inhibitor-treated conditions are not included in the figure for clarity but did not affect basal preproET-1 mRNA expression.

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).

View larger version (66K): [in this window] [in a new window]   Figure 8. Representative Northern blot analysis of total cellular RNA hybridized with endothelin-1 (ET-1) cDNA and GAPDH cDNA in endothelial cells exposed to known agonists A23187 (1 µmol/L for 2 hours) and transforming growth factor-ß (TGFß, 10 ng/mL for 2 hours). Incubations were also performed with herbimycin A (1 µmol/L for 18 hours), daidzein (6 µg/mL for 30 minutes), and genistein (6 µg/mL for 30 minutes) before exposure to the agonists. For comparison, lanes are shown for unstimulated control cells as well as for cells exposed to thrombin (4 U/mL for 120 minutes). Densitometric analysis data for preproET-1 mRNA, normalized to corresponding GAPDH signal expression, are shown underneath each lane. Data for inhibitor-treated conditions are not included in the figure for clarity but did not affect basal preproET-1 mRNA expression.

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).

View larger version (93K): [in this window] [in a new window]   Figure 9. Anti-phosphotyrosine immunoblot assay of SDS-polyacrylamide size-fractionated total cellular protein extracts, blotted with monoclonal PY20 anti-phosphotyrosine antibody, and visualized by chemiluminescence reagent in human umbilical vein endothelial cells exposed to thrombin (4 U/mL) in a time-dependent manner. For comparison, a lane is shown for unstimulated control cells (Contr). Migrations of prestained molecular masses are indicated at the left.

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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 TRAP^42-55 induced preproET-1 mRNA to comparable amounts, thus providing evidence for a receptor-mediated signaling event. However, TRAP^42-55 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, TRAP^42-55 failed to induce ET-1 peptide secretion in endothelial cells. The reasons for these differences between thrombin and TRAP^42-55 remain unclear. At present, thrombin receptor activation has not been shown to be reversible, and TRAP^42-55 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 TRAP^42-55 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 TRAP^42-55 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 TRAP^42-55 and its rapid degradation in the presence of aminopeptidases.³⁸ Other reports, showing data similar to ours, of the inability of TRAP^42-55 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 pp60^c-src activation in platelets.³⁰ Similar evidence has also been presented in rat and hamster fibroblasts; thrombin regulates p21^ras through activation of a heterodimeric G_i 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.

	Acknowledgments

 
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.

	References

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