Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, Matsumoto, 390-8621 Japan
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ABSTRACT |
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Effects of growth hormone (GH), insulin-like growth factor I (IGF-I), and endothelin-1 (ET-1) on endothelial cell migration and the underlying molecular mechanisms were explored using a human umbilical cord endothelial cell line, ECV304 cells, in vitro. Treatment of the cells with IGF-I or ET-1, but not GH, stimulated the cell migration. Interestingly, however, ET-1-induced, but not IGF-I-induced, migration of the cells was inhibited by GH. Both ET-1 and IGF-I caused activation of mitogen-activated protein kinase (MAPK) in the cells, and GH eliminated the MAPK activation produced by ET-1 but not that produced by IGF-I. On the other hand, migration of the cells was stimulated by protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate. ET-1 promoted PKC activity, and a PKC inhibitor, GF-109203X, blocked ET-1-induced cell migration. Although GH inhibited ET-1-induced cell migration and MAPK activity, it did not block ET-1-induced PKC activation. Thus ET-1 stimulation of endothelial cell migration appears to be mediated by PKC/MAPK pathway, and GH may inhibit the MAPK activation by ET-1 at the downstream of PKC.
protein kinase C; endothelin-1; Akt; phosphatidyl inositol 3'-kinase; mitogen-activated protein kinase
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INTRODUCTION |
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PATIENTS WITH ACROMEGALY or gigantism, who have high plasma levels of growth hormone (GH) and insulin-like growth factor I (IGF-I), often suffer from severe atherosclerosis (11). Acceleration of atherosclerosis is also a recent topic in studies of adult GH deficiency (15, 23). Increased atherogenesis in acromegaly may well be a consequence of the combined effect of hypertension, hyperinsulinemia, diabetes mellitus, and dyslipidemia, all of which are commonly seen in patients with acromegaly. In addition to these, GH stimulates production of a potent growth factor, IGF-I, in the liver (12, 21), which directly enhances cellular proliferation (5), even at the atherogenic sites (13). On the other hand, the acceleration of atherosclerosis in patients with adult GH deficiency suggests that GH and/or IGF-I possess an anti-atherogenic action. These seemingly puzzling phenomena could be explained if GH and IGF-I could be shown to have opposing effects at a certain stage or stages of atherosclerosis.
The cellular and subcellular processes in human atherosclerosis are not fully understood. However, these are generally considered complex processes consisting of endothelial cell injury (25), the transformation of macrophage and smooth muscle cells into the foam cells and their proliferation (27, 30), and eventually formation of atheroma (16). During the process of atherogenesis, endothelial cell migration also occurs (32), which may well be important in the initiation of angiogenesis and vasculogenesis at the site of ischemia and endothelial cell injury (31). Taking this information as background, we systematically evaluated the effects of GH and IGF-I on endothelial cell migration using human endothelial cells in vitro. We hypothesized that GH and IGF-I may have opposing effects at the level of endothelial cell migration, and we tried to delineate the underlying molecular mechanisms.
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MATERIALS AND METHODS |
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Cell Culture
A clonal human umbilical cord endothelial cell line, ECV304 cells, was obtained from Riken Cell Bank (Tokyo, Japan) and cultured in RPMI 1640 (GIBCO, Life Technologies) supplemented with 10% FBS (GIBCO) and 100 µg/ml crude endothelial cell growth factor (GIBCO) and 5 U/ml heparin (Novo Nordisk, Copenhagen, Denmark) at 37°C with 5% CO2.Chemicals
D-Glucose and phorbol 12-myristate 13-acetate (PMA) were purchased from Nakalai Tesque (Tokyo, Japan); LY-294002, GF-109203X, and PD-98059 were from Calbiochem (La Jolla, CA); human endothelin-1 (ET-1) was from Peptide Institute (Osaka, Japan); IGF-I and epidermal growth factor (EGF) were from GIBCO; and human growth hormone (hGH) was from Sumitomo Pharmaceutical (Tokyo, Japan). Anti-mitogen-activated protein kinase (Cell Migration Assay
Cell migration assay was performed as previously described (17) with minor modifications (29). The migration was determined in 24-well plates with 8-µm pore size cell culture inserts. Sub-confluent ECV304 cells were harvested with trypsin/EDTA and then washed and resuspended, at 1 × 106 cells/ml, in Ham's F12K (GIBCO) with 1% FBS, and a 0.2-ml aliquot was placed onto each insert. The inserts were then placed in the wells containing 0.7 ml serum-free Ham's F12K with type 1 collagen (Sigma, 3 mg/ml). The cells were incubated for 4 h allowing migration through the 8-µm pores of the inserts, and the effects of various agents were determined. Thus exposure to the test substance began at the time the cells were aliquoted onto the insert. When the effect of the test substance was evaluated, both top and bottom wells were filled with the same solution containing the same test substance. Therefore, we have evaluated chemokinesis (undirected movement) in this study. Cell migration in the absence of test substance was always determined in parallel, and this was taken as the control.The migration was quantified as follows. All nonmigrated cells were removed from the upper surface of the insert membrane with a scraper, and the migrated cells, attached to the lower surface, were fixed and stained with May-Grunwald and Giemsa solutions. The number of stained cells was counted with a microscope at a magnification of ×200. The following reagents were used at the concentrations indicated: a PKC inhibitor, GF-109203X (200 nM); a PKC agonist, PMA (100 nM); a MAPK/ERK kinase (MEK) inhibitor, PD-98059 (10 µM); a phosphatidyl inositol 3'-kinase (PI3-K) inhibitor, LY-294002 (200 nM); ET-1 (1 µM); EGF (200 nM); GH (40 pM); insulin (100 nM); and glucose (35 mM).
GH Binding Assay
In this experiment, Krebs-Ringer bicarbonate/phosphate buffer containing 125 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 10 mM sodium phosphate, 25 mM sodium bicarbonate, and 5 mM HEPES, pH 7.6, and 0.1% BSA, with or without various concentrations of unlabeled GH, was used. Two days prior to use, the cells were plated and placed in a serum-free medium for 12 h. Then, the cells (106 cells/dish) were incubated with 125I-labeled GH (1 µCi/dish, New England Nuclear, DuPont) at 10°C for 1 h. At the end of incubation, the cells were washed three times with ice-cold Krebs-Ringer bicarbonate/phosphate buffer and lysed by addition of 1 ml 0.1 N NaOH. The lysate was transferred to the tube, and the radioactivity was counted. Binding of 125I-GH in the presence of 10 nM GH was defined as nonspecific binding.Western Blot Analysis
Whole cell extracts were prepared by exposing the cells to lysis buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, and 1.5 µM pepstatin) for 1 h at 4°C. The resultant cell extracts were electrophoresed as previously described (34) and subjected to Western blotting using antibodies againstMeasurement of PKC Activity
The cytosol fraction was obtained by the ultracentrifugation as previously described (8) with minor modifications. ECV304 cells were removed from the plates by rubber policeman and homogenized in ice-cold buffer containing 20 mM Tris · HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 1 mM NaHCO3, 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µg/ml aprotinin, and 1.5 µM pepstatin. Then the homogenate was centrifuged at 5,000 g for 30 min at 4°C, and the supernatant was centrifuged at 100,000 g for 60 min at 4°C. The pellet was collected as the membrane fraction. The activity of PKC was measured by MESACUP protein kinase assay kit. This kit is based on enzyme-linked immunosorbent assay (ELISA) that utilizes a synthetic polypeptide as PKC substrate and a monoclonal antibody recognizing phosphorylated form of the peptide. Protein was measured by Pierce bicinchoninic acid (BCA) protein assay kit (Rockford, IL) using BSA as a standard.Statistical Analysis
Statistical significance of difference was evaluated using ANOVA with Fisher's PLSD test (StatView; SAS Institute, Cary, NC). A P <0.05 was considered statistically significant. ![]() |
RESULTS |
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Investigation of the Cell Migration
Effects of GH and IGF-I.
GH alone did not increase the number of migrating cells even at 40 pM
(Fig. 1A). This concentration
of GH showed >90% displacement in GH binding assay (Fig.
2A). In contrast, IGF-I
significantly increased the migration of ECV304 cells: the minimum
effective concentration was 1 nM, the EC50 was 3 nM, and
the maximum effect was observed at 30 nM (Fig. 1B).
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Effects of EGF, ET-1, PMA, and high glucose.
As shown in Fig. 3A, another
growth factor, EGF, and a vasoconstrictor, ET-1, also increased the
number of migrating cells. Previously, we observed that a high
concentration of glucose increased the number of migrating cells by
activation of PKC (29). As shown in Fig. 3B, we
confirmed that result in this experiment. Furthermore, PMA, a PKC
agonist, also stimulated the cellular migration (Fig. 3B).
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Effects of a PKC inhibitor, GH, and a combination thereof on the
growth factor-induced cell migration.
As shown above, activation of PKC led to stimulation of ECV304 cell
migration. Thus we tested a PKC inhibitor, GF-109203X, to see whether
this is also the mechanism involved in the growth factor-induced
cell migration. GF-109203X effectively inhibited the migration
induced by ET-1 or high glucose but not that induced by EGF or IGF-I
(Fig. 4A). These results
suggested the involvement of PKC in ET-1- and high-glucose-induced
migration but not in IGF-I- and EGF-induced migration.
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Effects of PI3-K and MEK inhibitors on the IGF-I- and ET-1-induced
cell migration.
IGF-I generally activates the signaling through both PI3-K and Ras-MAPK
pathways, and downstream molecules thereof (1). To
identify the mechanism for stimulation of cell migration by IGF-I,
inhibitors of PI3-K and MEK were tested. LY-294002, a potent PI3-K
inhibitor (4), completely eliminated the IGF-I-induced cell migration. However, PD-98059, a selective MEK inhibitor
(18), did not attenuate the IGF-I effect at all (Fig.
6), suggesting that it is PI3-K rather
than MEK which is involved in the IGF-I-induced cell migration. In
contrast, PD-98059 clearly suppressed ET-1-induced cell migration (Fig.
5A), indicating MEK mediation of the ET-1 effect.
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Investigation of the Intracellular Signaling Molecules
Effects of GH, PD-98059, and GF-109203X on activation of
intracellular signaling molecules by IGF-I and ET-1.
PD-98059, a MEK inhibitor, clearly blocked MAPK activation induced by
IGF-I (Fig. 7A). In Fig.
7B, IGF-I-induced phosphorylation of Akt in the presence of
PD-98059 was slightly stronger than in the absence of PD-98059.
However, based on the data obtained in three independent experiments,
we concluded that IGF-I-induced activation of Akt is not affected by
PD-98059; by densitometry, phosphorylation of Akt by IGF-I in the
presence of PD-98559 was 98 ± 28% (n = 3) of
that by IGF-I alone. GH alone only marginally activated Akt and MAPK
(Fig. 7, A and B). Whereas GH and GF-109203X, a
potent PKC inhibitor, inhibited ET-1-induced MAPK activation but had no
effect at all on IGF-I-induced MAPK and Akt activation (Fig. 7,
A and B).
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Effect of GH on ET-1-induced PKC activation.
The effect of GH on the ET-1 activation of PKC was examined by directly
measuring PKC activity. As shown in Fig.
8, treatment of the cells with ET-1
promoted activation of PKC, which was not affected by GH. Although it
was reported that GH activates PKC in many cells (2), GH
per se did not cause activation of PKC in ECV304 cells (Fig. 8).
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DISCUSSION |
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In this study, we systematically analyzed the effects of GH and various growth/vascular factors on the migration of ECV304 cells and investigated the underlying molecular mechanisms. First, it was observed that IGF-I, EGF, and ET-1 all stimulated migration of the cell, but GH itself had no effect. Nevertheless, GH specifically inhibited the ET-1 effect on the endothelial cell. Second, with respect to mediation of the migration, IGF-I utilizes the PI3-K branch, but not the Ras-MAPK branch, in the cell line. This action of IGF-I was unaffected by GH. In contrast, ET-1, like vascular endothelial growth factor (VEGF) (33), may activate MAPK through activation of PKC in human vein umbilical endothelial cells. GH inhibition of ET-1 signaling takes place downstream of PKC, not at the level of the ET-1 receptor, because GH does not interfere with ET-1-induced activation of PKC but selectively attenuates ET-1-induced MAPK activation.
We were unable to delineate mediation of GH inhibition at downstream of PKC in this study. However, it is likely that Janus kinases (JAK) mediate the inhibitory action of GH (2). Namely, upon binding of GH to its receptor, JAK kinases became activated and form complexes with GH receptor subunits. JAK kinases phosphorylate the GH receptor and tyrosine residues of their own. Existence of a JAK/STAT signaling pathway was recently demonstrated in ECV304 cells (3). GH might interfere with activation of MAPK by ET-1 downstream of PKC (see below).
Several previous data are worth commenting upon here. First, involvement of PKC in GH action is disputed. It has been suggested that PKC activation is involved in GH stimulation of lipogenesis, c-fos expression, binding of nuclear protein to C/EBP oligonucleotide, and elevation of cytosolic free Ca2+ concentration (2). On the other hand, Goodman et al. (10) found that lipolytic activity of GH might not be mediated by PKC in adipocytes. We did not see activation of PKC or migration (an expected consequence of PKC activation) in ECV304 cells. By using the method employed here, activities of Ca2+-dependent PKC isoforms but not Ca2+-independent PKC isoforms can be detected (19). Therefore, an active role for conventional PKC in GH signaling is most unlikely in this cell line. Involvement of Ca2+-independent PKC isoforms cannot be ruled out.
Second, as far as we are aware, this is the first demonstration of the negative effects of GH on ET-1 down-signaling and its cellular action, namely, induction of endothelial cell migration. In fact, the vasopressor action of ET-1 was well known, but activation of endothelial cell migration by the peptide has not been reported before. The involvement of PKC and MAPK in the signal transduction of ET-1 was previously suggested by Schiffrin and Touyz (28) in endothelial cells. More recently, VEGF-induced activation of MAPK via PKC was demonstrated in a human umbilical cord endothelial cell line (33). Because MEK inhibitor restrained ET-1-induced cell migration in our study, GH, downstream of PKC, might be acting at this stage to interfere with the ET-1 action.
Third, IGF-I-induced migration has been reported in various endothelial cells (6, 14, 22, 26), and its mediation has not been thought to be by PKC (7). The role of MAPK in IGF-I down-signaling is disputed. In some studies (9, 24), mediation by MAPK of IGF-I action was demonstrated. However, Manes et al. (20) recently reported that this is not the case in human breast adenocarcinoma cells. Our data regarding ECV304 cells are compatible with the conclusion of Manes et al. (20)
As we observed, the GH-IGF-I axis plays a complex role in the migration of endothelial cells. Provided that endothelial cell migration can be regarded as an index of vascular reaction in vivo, our data shed some light on the atherogenesis in acromegaly and adult GH deficiency. In acromegaly, together with other metabolic derangements such as diabetes, dyslipidemia, hyperinsulinemia, and hypertension, high plasma concentration of IGF-I will directly enhance atherogenesis. Increased levels of GH itself do not have either a positive or negative effect on atherogenesis. Whereas, in adult GH deficiency, because of the abnormally low concentration of GH in the plasma, the inhibitory effect of GH on the endothelial activation by ET-1 and most likely by other vasoactive substances, as well, is lost, thereby accelerating atherosclerosis. Under these conditions, the concentration of plasma IGF-I is also low, thus excluding any possibility of the involvement of this peptide in the acceleration of atherosclerosis.
In this study, we investigated the role of the GH-IGF-I axis in endothelial cell migration and found that GH and IGF-I play important roles in the downregulation and upregulation of endothelial cell migration, respectively. GH suppresses ET-1-induced endothelial cell migration by suppression of MAPK at the downstream of PKC. On the other hand, GH simulates the migration indirectly through production of IGF-I in vivo. The results suggest the complex role of GH as a homeostatic balancer in atherogenesis. Although these mechanisms are probably important in atherogenesis, there is a limitation in our approach, because we studied only undirected motility of endothelial cells. Further studies are needed to elucidate the whole picture of the cellular and molecular mechanisms of GH and IGF-I action.
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ACKNOWLEDGEMENTS |
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We thank Professor D. E. Ruzicka for editorial assistance.
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FOOTNOTES |
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This work was supported by a Ministry of Education, Science and Culture (Japan) Grant-in-Aid for Scientific Research (to K. Yamauchi).
Address for reprint requests and other correspondence: K. Yamauchi, Dept. of Aging Medicine and Geriatrics, Shinshu Univ. School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan (E-mail: keishi{at}hsp.md.shinshu-u.ac.jp).
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.
Received 22 February 2000; accepted in final form 18 December 2000.
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