1Cardiovascular Research Center and 6Department of Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; 2Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; 3Department of Pathology, Wakayama Medical University, Wakayama 641-8509, Japan; 4Pacific Northwest Research Institute, Seattle 98112; and 5Department of Medicine, University of Washington, Seattle, Washington 98195
Submitted 9 October 2003 ; accepted in final form 20 May 2004
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ABSTRACT |
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epidermal growth factor receptors; ErbB2; migration; signal transduction
BTC, a novel member of the EGF family, was originally isolated as a growth-promoting factor in conditioned medium derived from a mouse pancreatic beta cell line (29). BTC is expressed in a wide range of tissues, including kidney, liver, and small intestine, and is particularly high in the pancreas (7). Interestingly, BTC was shown to be a potent mitogen for cultured VSMCs (29). BTC precursor protein is expressed in intimal and medial VSMCs of human atherosclerotic lesions (32). However, the exact function of BTC in mediating vascular remodeling is not completely characterized, and the activation of ErbB receptors and postreceptor signaling by BTC in VSMCs is poorly defined. In light of this, we hypothesized that there is a plausible participation of this new EGF ligand and its signaling in vascular pathological conditions. Therefore, the aim of the present study was to characterize the signal transduction of BTC in mediating growth and migration of VSMCs.
In this study, we demonstrate that BTC activates EGFR, ErbB2, and their postreceptor signal transduction pathways, leading to growth and migration responses in VSMCs. Moreover, we show the presence and processing of endogenous BTC in VSMCs, suggesting that BTC acts as an autocrine and/or paracrine growth factor in mediating vascular remodeling.
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MATERIALS AND METHODS |
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Cell culture.
VSMCs were prepared from thoracic aorta of Sprague-Dawley rats by the explant method. All animal procedures conformed to the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. Subcultured cells from passages 312 were used and showed 99% positive immunostaining with smooth muscle -actin antibody (9). For the experiments, VSMCs at 8090% confluence were used after serum depletion with 0% serum Dulbecco's modified Eagle's medium for 23 days.
Immunoprecipitation. After stimulation at 37°C, cells were lysed with ice-cold immunoprecipitation buffer as previously described (9). The cell lysates or the conditioned medium was centrifuged, and the supernatant was immunoprecipitated with the antibody and protein A/G plus agarose at 4°C for 16 h.
Western blotting. Cell lysates or immunoprecipitation lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electrophoretically transferred to a nitrocellulose membrane as previously described (9). The membranes were then exposed to primary antibodies overnight at 4°C. After incubation with the peroxidase-linked secondary antibody for 1 h at room temperature, immunoreactive proteins were visualized with ECL reagent (Amersham Life Sciences).
DNA synthesis. DNA synthesis was assessed by incorporation of 3H-labeled thymidine into cells as previously described (21). In brief, quiescent VSMCs were incubated with BTC for 24 h and 1 µCi of [3H]thymidine was added for the last 8 h. Trichloroacetic acid-insoluble radioactivity was measured in a liquid scintillation counter.
Proliferation assay. Cell proliferation was measured by using the CellTiter 96 AQueous cell proliferation assay kit (Promega) according to the manufacturer's protocol. In brief, quiescent VSMCs in 96-well plates were pretreated with or without inhibitors for 1 h and then stimulated with BTC (20 ng/ml) for 48 h. After incubation with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt solution provided by the kit, viable cells were determined at 490-nm absorbance with a 96-well plate reader.
Migration assay. Cell migration was performed with the Boyden chamber method as previously described (26). Briefly, the lower well of the chemotaxis chamber was filled with 600 µl of serum-free medium that contained BTC (20 ng/ml) with or without inhibitors. An 8-µm polycarbonate filter was placed between the upper and lower chambers. VSMCs were trypsinized and resuspended in serum-free medium to a concentration of 105 cells/100 µl. This volume was then added to the upper chamber. After incubation for 8 h at 37°C in a 5% CO2 incubator, filters were removed and the cells on the top surface of the membrane were removed with a cotton swab. The membranes were washed with phosphate-buffered saline, fixed with methanol, and stained with Giemsa stain. Cells were counted with the use of a light microscope.
RT-PCR. Expression of BTC mRNA was analyzed by the RT-PCR method as previously described (21). Primers for BTC were 5'-GTCCTGGGTCTTGTGATTC-3' and 5'-GAAGAGGATGACAGCAGGT-3', which correspond to bases 106124 and 466484 of the rat BTC sequence (31), respectively. By using rat BTC cDNA (31) as a template, we have confirmed that the BTC primers used in this study yield an expected single band (376 bp). The amplification was done with PCR System 2700 (Applied Biosystems) with a Gene Amp RNA PCR core kit (Roche). Reaction products were resolved by electrophoresis through 2% agarose gels and were visualized with a laser image analyzer.
Statistical analysis. Data were analyzed by Student's t-test. Means ± SE were determined with a significance level of P < 0.05. The results shown in blots are representative of at least three separate experiments using at least two distinct batches of VSMCs.
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RESULTS |
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DISCUSSION |
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The biological actions of EGF family ligands are mediated through a member of receptor tyrosine kinases, ErbB receptors (34). Our results showed that BTC stimulates tyrosine phosphorylation of various proteins in VSMCs. Among these proteins, 170-kDa proteins were identified as EGFR and ErbB2. Although we have not characterized the other tyrosine-phosphorylated proteins (110120, 7075, and 5060 kDa) induced by BTC, we assume that these proteins represent substrate and adaptor proteins of EGFR and ErbB2, such as Src (60 kDa), Cbl (120 kDa), SHP-2 (66 kDa), and Shc (52 and 46 kDa). As shown in Fig. 1C, EGFR phosphorylation by BTC occurred at a maximum concentration of 20 ng/ml, which is compatible with a recent study reported by Shin et al. (28) in VSMCs. Also, our results show that EGFR phosphorylation by BTC was equivalent to the phosphorylation induced by EGF or HB-EGF, suggesting similar potency of BTC in its growth-promoting effects on VSMCs.
Recent studies suggested that BTC is able to induce all possible combinations of homo- and heterodimer ErbB receptors, which could be cell type and tissue dependent (7). In this study, we showed that BTC stimulates marked phosphorylation of ErbB2 at three intrinsic tyrosine residues in VSMCs. Among these, Tyr1112 and Tyr1248 are considered as autophosphorylation sites, indicating that BTC stimulates the kinase activity of ErbB2. Because ErbB2 is known to heterodimerize with all other ErbB receptors (34), the association of EGFR and ErbB2 observed in this study suggests a heterodimerization of EGFR and ErbB2 induced by BTC in VSMCs. These data suggest that, in addition to its critical involvement in heart development (19) and cancer cell growth (1), ErbB2 may also play an important pathological role in vascular remodeling. AG-1478 is a well-recognized EGFR/ErbB1-selective kinase inhibitor. In the present study, the EGFR/ErbB1 kinase inhibitor AG-1478, which acts by competing with ATP (11), completely blocked ERK1/2 activation, DNA synthesis, and migration of VSMCs induced by BTC. However, because AG-1478 was reported to inhibit ErbB2 function by inducing an inactive heterodimer of EGFR/ErbB2 (2, 11), we could not distinguish the actions of these two receptors in mediating BTC function in VSMCs at this point. Shin et al. (28) reported that BTC induces tyrosine phosphorylation of ErbB3 and ErbB4 in addition to ErbB1 and ErbB2 in human aortic VSMCs in culture. Although we could detect transcripts of ErbB3 and ErbB4, we could not detect their protein expression in our VSMCs (Dempsey PJ and Eguchi S, unpublished observation). This may involve a difference in species; however, this may also indicate that ErbB3 and ErbB4 could be dispensable for BTC-induced growth and migration of VSMCs. The functional significance of each ErbB receptor in vascular diseases requires further examination.
Regarding postreceptor signal transduction, we showed that BTC activates ERK1/2, Akt, and p38 MAPK in VSMCs. Activation of each of these kinases by various agonists, such as angiotensin II, has been shown to be critically involved in growth promotion and/or migration of cultured VSMCs (8, 13, 25, 27, 33). In human aortic VSMCs, BTC-induced DNA synthesis and cell cycle progression were markedly inhibited by a phosphatidylinositol 3-kinase inhibitor, wortmannin, but not by an ERK inhibitor, PD-98059 (28), indicating a dominant role of phosphatidylinositol 3-kinase/Akt pathway in mediating VSMC growth by BTC. In contrast, our data showed a partial involvement of Akt as well as ERK in BTC-induced proliferation of rat VSMCs. The difference in these results might involve an Akt-independent growth pathway (ERK and/or other phosphatidylinositol 3-kinase downstream) operated by BTC in rat VSMCs as well as different responses to the PD-98059 concentration utilized. In addition, we have further addressed postreceptor signaling mediating VSMC migration induced by BTC. Our data indicate the requirement of ERK, Akt, as well as p38 MAPK to fully establish migration of VSMCs induced by BTC. This is consistent with the requirement of ERK, p38 MAPK, and phosphatidylinositol 3-kinase in VSMC migration induced by platelet-derived growth factor (16, 35).
In the present study, we demonstrated a transcript of membrane-anchored BTC (proBTC) by RT-PCR and release of mature BTC into the conditioned medium after angiotensin II stimulation. Together with the expression of BTC in human atherosclerotic lesions (28, 32), this result suggests that endogenous BTC produced from VSMC may be an important growth and migration factor involved in vascular disease states. It is now recognized that several G protein-coupled receptor ligands exert their growth-promoting action largely through the "trans"-activation of the EGFR (5, 9). In this signaling, metalloprotease-dependent cleavage of membrane-anchored EGF family ligand appears to be crucial (23). Our group recently reported that there is a major contribution of a metalloprotease-dependent EGFR ligand production in EGFR transactivation in VSMCs stimulated by angiotensin II (8) and reactive oxygen species (10). Very recently, EGFR transactivation by cleaved BTC was reported in pancreatic beta cells (4). Together, our results suggest that BTC could be a ligand involved in the EGFR/ErbB transactivation in VSMCs.
In summary, this study found that BTC promotes proliferation and migration of VSMCs by activating EGFR/ErbB2 and downstream serine/threonine kinases. Together with the evidence of endogenous BTC expression and processing by angiotensin II in VSMCs, our findings suggest that production and signal transduction of BTC may mediate a critical pathological step of vascular disease. Therefore, our data provide a new mechanistic insight by which several risk factors induce vascular remodeling by possibly inducing BTC-mediated EGFR/ErbB2 signaling pathways.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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