Endothelin-1 Stimulates DNA Synthesis of Vascular... : Journal of Cardiovascular Pharmacology (original) (raw)
Endothelin-1(ET-1) is a potent vasoconstrictor peptide, originally isolated from the supernatant of cultured porcine endothelial cells (1). Subsequent cDNA cloning of the human genomic library uncovered three ET peptides (ET-1, ET-2, and ET-3) (2). Their actions are mediated by at least two receptor subtypes, ET-1-selective type A (ETA) and non-isopeptide-selective type B (ETB)(3,4). These receptors contain seven transmembrane domains common to the superfamily of GTP-binding protein (G-protein)-coupled receptors.
We and others have previously shown that vascular smooth-muscle cells (VSMCs) predominantly express ETA receptors, which not only mediate vasoconstriction but also promote proliferation of VSMCs (5,6). We have further demonstrated that activation of the Gq-coupled ETA receptor in rat VSMCs results in formation of inositol 1,4,5-triphosphate (IP3), leading to intracellular Ca2+ mobilization (5). In addition, Komuro et al. (7) have reported that ET-1 induced a rapid and transient increase in the c-fos and c-myc mRNA levels in VSMCs. However, the signal transduction pathway of ET-1 that mediates its mitogenic action remains unclear. In the present study we examined the possible role of epidermal growth factor receptor (EGFR) in ET-1-induced mitogen-activated protein (MAP) kinase activation, c-Fos expression and DNA synthesis in rat VSMCs.
MATERIALS AND METHODS
Compounds
Dulbecco's modified Eagle's medium (DMEM) was purchased from Life Technologies (Rockville, MD, U.S.A.), synthetic ET-1 from Peptide Institute (Osaka, Japan), AG1478 from Calbiochem-Novabiochem(La Jolla, CA, U.S.A.), and [3H]thymidine (specific activity 6.7 Ci/mmol) from New England Nuclear (Boston, MA, U.S.A.).
Cell culture
Rat VSMCs were prepared from the thoracic aorta of 12-week-old male Sprague-Dawley rats(Charles River Laboratories, Wilmington, DE, U.S.A.) by the explant method and were cultured in DMEM containing 10% FCS at 37°C in a humidified atmosphere of 95% air-5%CO2 as described (6). Quiescent VSMCs (5-15th passages) after 72-h serum-starvation were used in the following experiments.
MAP kinase activity
Quiescent VSMCs pretreated with or without AG1478 (2.5 × 10−8_M_-2.5 × 10−7_M_) for 30 min were stimulated with ET-1 (10−7_M_) at 37°C in serum-free DMEM for 5 min. The reaction was terminated by the replacement of medium with ice-cold lysis buffer. After brief sonication, the samples were centrifuged and the supernatants were assayed for MAP kinase activity using an assay kit (Amersham International, Poole, U.K.).
Western blotting
Western blotting was performed as previously described (8). Quiescent VSMCs pretreated with or without AG1478 (2.5 × 10−7_M_) for 30 min were stimulated with ET-1 (10−7_M_) at 37°C for the indicated times under serum-free conditions. The reaction was terminated by the replacement of medium with 100 μl of SDS-polyacylamide gel electrophoresis (PAGE) buffer. After brief sonication, samples were boiled for 5 min at 95°C and centrifuged, and aliquots of the supernatants were subjected to 10% SDS-PAGE. Proteins in the gel were transferred to a nitrocellulose membrane by electroblotting. The membrane was treated with anti-rabbit polyclonal c-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). After incubation, immunoreactive proteins were detected by the ECL system (Amersham International).
DNA synthesis
DNA synthesis was assessed by incorporation of [3H]thymidine into cells as described (6). In brief, the quiescent VSMCs pretreated with or without AG1478 (2.5 × 10−8_M_-2.5 × 10−7_M_) for 30 min were incubated with ET-1 (10−7_M_) for 20 h, after which 1 μCi [3H]thymidine was added and the cells were further incubated for 4 h. After completion, trichloroacetic acid-insoluble radioactivity was measured by a liquid scintillation counter.
RESULTS
ET-1(10−7_M_) induced a rapid and transient activation of MAP kinase, which peaked at 5 min and declined at 10 min. AG1478 dose-dependently (2.5× 10−8_M_-2.5 × 10−7_M_) inhibited ET-1-induced MAP kinase activation (data not shown).
As shown inFig. 1, ET-1 (10−7_M_) markedly induced expression of c-Fos proteins (∼130-140 kDa) which was detected within 0.5 h, peaked at 1 h, and was sustained up to 4 h. Pretreatment with AG1478 (2.5 × 10−7_M_) inhibited c-Fos protein expression induced by ET-1.
Effect of AG1478 on c-Fos protein expression induced by ET-1 in cultured rat VSMCs. After treatment with or without AG1478 (2.5× 10−7 M) for 30 min, cells were stimulated with ET-1(10−7 M) for the indicated times. Western blot analysis was performed using an anti-c-Fos antibody.
As shown in Fig. 2, pretreatment with AG1478 for 30 min also dose-dependently (2.5 × 10−8_M_-2.5 × 10−7_M_) inhibited ET-1-induced[3H]thymidine incorporation, whereas it had no effect on the basal level.
Effect of AG1478 on ET-1-induced DNA synthesis in cultured rat VSMCs. After treatment with or without AG1478 (2.5× 10−8 _M_-2.5 × 10−7 M) for 30 min, cells were incubated with or without ET-1 (10−7 M) for 20 h. [3H]Thymidine incorporated during 4 h was measured. Each point represents the mean ± SEM (n = 3).
DISCUSSION
In addition to its potent vasoconstrictor effect, ET-1 has been shown to stimulate DNA synthesis in VSMCs (6,7). The present study demonstrated that ET-1 stimulated MAP kinase, c-Fos expression, and DNA synthesis of rat VSMCs through transactivation of EGFR.
In the present study, ET-1 induced the expression of c-Fos proteins in a time-dependent manner, as demonstrated by Western blot analysis using an anti-c-Fos antibody. The time course of c-Fos protein expression appears to be comparable to that of ET-1-induced c-fos mRNA expression, as determined by Northern blot analysis (7). However, the apparent molecular weights (∼130-140 kDa) of immunoreactive c-Fos proteins observed in this study were larger than that of c-Fos protein (62 kDa) (9). This may be due to the ubiquitinylated protein after its phosphorylation (10) or to other unidentified c-Fos family proteins that crossreacted with the anti-c-Fos antibody.
This study confirmed that ET-1 activates MAP kinase in VSMCs (11) and further revealed that ET-1-induced MAP kinase activation is inhibited by an inhibitor of EGFR kinase (AG1478). It is generally recognized that MAP kinase phosphorylates transcription factor Elk1, thereby leading to c-fos gene expression via a serum-responsive element (SRE) in its promoter region (12). Therefore, we can speculate that ET-1 may activate MAP kinase, c-Fos protein expression, and DNA synthesis through transactivation of EGFR in rat VSMCs. However, the partial inhibition of c-Fos expression by AG1478 suggests that c-Fos expression by ET-1 may not be solely explained by transactivation of the receptor.
In this study we have also shown that pretreatment with AG1478 completely blocks ET-1-induced DNA synthesis. We have previously demonstrated that ET-1 stimulates DNA synthesis via the Gq-coupled ETA receptor in cultured rat VSMCs (5). We have recently reported that p21Ras and MAP kinase activation by angiotensin II are mediated through a tyrosine kinase that exists downstream of the Gq/phospholipase C pathway (8). Quite recently, Daub et al. (13) have demonstrated that the G-protein-coupled receptor agonists such as ET-1, thrombin, and lysophosphatidic acid, induced tyrosine phosphorylation of the EGFR in Rat-1 fibroblasts, and that treatment with AG1478 or transfection of a dominant negative EGFR mutant suppressed the MAP kinase activation, c-fos gene expression, and DNA synthesis induced by these agonists. Taken together, our results suggest that transactivation of the EGFR is the critical point of convergence in the growth promoting cascade shared by Gq-coupled receptors. A possible signal transduction of ET-1-induced growth of VSMCs via EGFR transactivation can be postulated, as illustrated in Fig. 3.
Possible molecular mechanism of VSMC growth stimulated by ET-1. ET-1 induces MAP kinase activation and c-Fos expression, and promotes proliferation of VSMCs via transactivation of the EGF receptor (EGFR). Gq-coupled ETA receptor transactivates the EGFR, resulting in recruitment of adaptor proteins (Shc, Grb2, and Sos). Then, Sos catalyzes the exchange of GDP to GTP on p21Ras, leading to activation of the MAP kinase cascade and c-Fos expression, and ultimately promoting DNA synthesis.
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Keywords:
Endothelin-1; Epidermal growth factor receptor; c-Fos; DNA synthesis
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