1Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, and 2Augusta Veterans Affairs Medical Center, Augusta, Georgia 30912
Submitted 21 November 2002 ; accepted in final form 21 April 2003
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ABSTRACT |
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receptor; splice variants
Although GIP receptors are widely distributed in the endothelium (28), their role in this tissue is not completely clear. We have recently reported that multiple GIP receptor splice variants are present in different endothelial cell (EC) types (31). Human umbilical vein endothelial cells (HUVEC) were found to contain two GIP receptor splice variants, whereas a spontaneously transformed umbilical vein EC line, ECV 304 cells, had four receptor splice variants. Although GIP increased intracellular calcium in both EC types, GIP activated cAMP-dependent protein kinase in the HUVEC but not in the ECV 304 cells, suggesting that the splice variants could be differentially linked to signaling pathways (31). Interestingly, infusion of GIP has been shown to have different effects on blood flow in vivo; i.e., it increases portal venous flow and decreases hepatic artery blood flow (13), suggesting that GIP has distinct vasoactive properties.
In an attempt to shed more light on GIP's role in the endothelium, GIP effects on vascular EC proliferation (by use of [3H]thymidine incorporation as a measure) and the underlying mechanism of these effects were investigated. Our present results demonstrate that GIP modulates proliferation in HUVEC through stimulation of endothelin (ET)-1 synthesis and release and subsequent activation of the endothelin B receptor (ETBR) subtype. Although GIP also stimulates [3H]thymidine incorporation in ECV 304 cells, this effect does not seem to mediated by ET-1. In view of the differences in cellular responses to GIP in HUVEC vs. ECV 304, linkage of GIP receptor splice variants to different transducers in these two cell types might in fact be responsible for the observed differences.
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MATERIALS AND METHODS |
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GIP, ET-1, BQ-788, and BQ-610 were from Bachem (Torrance, CA). The 125I-labeled ET-1 radioimmunoassay (RIA) kit was purchased from Peninsula Labs (San Carlos, CA). [Methyl,1',2'-3H]thymidine was from Amersham (Arlington Heights, IL).
Cell Culture
For these studies, we used normal HUVEC (Clonetics, San Diego, CA), as well as the immortalized HUVEC cell line ECV 304 (ATCC, Gaithersburg, MD) (26). Cells were grown to confluence in endothelial growth medium (EGM; Clonetics) or Medium 199 (Whittaker Biologic Products, San Diego, CA), supplemented with 10% FCS (vol/vol; HyClone Laboratories, Logan, UT), penicillin (100 U/ml), streptomycin (100 mg/ml), and amphotericin B (3 mg/ml).
[3H]Thymidine Incorporation Assay
EC were seeded in 6-well plates at 2 x 105/well and allowed to attach and reach 80% confluence. Cells were then placed in a modified Krebs-Ringer bicarbonate (KRB) buffer (containing in mM: 25 sodium bicarbonate, 120 sodium chloride, 4 potassium chloride, 1.2 magnesium sulfate, 1.2 sodium bisphosphate, 5.6 dextrose, 1.25 calcium chloride, and 2 mg BSA/ml) overnight. The KRB buffer was then changed, and the appropriate agonist was added for an additional 24 h. ET receptor antagonists were incubated for 15 min before the addition of agonist. During the last 6 h of incubation, 1 µCi/well of [3H]thymidine was added. Cells were then washed three times with phosphate-buffered saline, treated twice with 5% TCA (ice cold) for 15 min, washed once with water, and solubilized in 0.3 N sodium hydroxide. The cell-associated radioactivity was then determined by liquid scintillation counting.
Microarray Analysis
T7-based RNA amplification. Ten micrograms of total RNA were converted into double-stranded cDNA (ds-cDNA) by use of the SuperScript Choice System (GIBCO-BRL Life Technologies) with an oligo(dT) primer containing a T7 RNA polymerase promoter (Genset). After second-strand synthesis, the reaction mixture was extracted with phenol-chloroform-isoamyl alcohol, and ds-cDNA was recovered by ethanol precipitation.
Labeling, hybridization, and scanning. In vitro transcription was
performed on the above ds-cDNA with the Enzo RNA transcript labeling kit.
Biotin-labeled cRNA was purified by use of an RNeasy affinity column (Qiagen)
and fragmented randomly to sizes ranging from 35 to 200 bases by incubating at
94°C for 35 min. The hybridization solutions contained 100 mM MES, 1 M
Na+, 20 mM EDTA, and 0.01% Tween 20. The final concentration of fragmented
cRNA was 0.05 µg/µl in the hybridization solution. Target for
hybridization was prepared by combining 40 µl of the fragmented transcript
with sonicated herring sperm DNA (0.1 mg/ml), BSA, and 5 nM control
oligonucleotide in a buffer containing 1.0 M NaCl, 10 mM Tris · HCl (pH
7.6), and 0.005% Triton X-100. Target was hybridized for 16 h at 45°C to a
set of oligonucleotide arrays (HG U133A; Affymetrix, Santa Clara, CA). Arrays
were washed at 50°C with stringent solution and then at 30°C with
nonstringent solutions. Arrays were then stained with
streptavidin-phycoerythrin (Molecular Probes). DNA chips were read at a
resolution of 3 µm with a Hewlett-Packard GeneArray Scanner and were
analyzed with the GENECHIP software (Affymetrix). Detailed protocols for data
analysis of Affymetrix microarrays and extensive documentation of the
sensitivity and quantitative aspects of the method have been described
(14). Briefly, each gene is
represented by the use of 20 perfectly matched (PM) and mismatched (MM)
control probes. The MM probes act as specificity controls, which allows the
direct subtraction of both background and cross-hybridization signals. The
number of instances in which the PM hybridization signal is larger than the MM
signal is computed along with the average of the logarithm of the PM-to-MM
ratio (after background subtraction) for each probe set. These values are used
to make a matrix-based decision concerning the presence or absence of an RNA
molecule. Comparison analysis between control and experimental animals was
made with Affymetrix software. Further analysis for functional classification
was carried out using GeneSpring (Silicon Genetics, Redwood, CA).
ET-1 Measurements
ET-1 measurements were performed as previously described (12). Briefly, EC were grown in 6-well plates, placed in 0.1% FCS overnight before use, and then incubated for an additional 20 h with the appropriate agonist. The cell culture supernatants were removed, and ET-1 contents were measured with a commercially available RIA kit (Peninsula Labs).
Statistics
Results are expressed as means ± SE. Experiments were performed in triplicate except where noted. Data were analyzed with either ANOVA or unpaired t-tests, where appropriate, with a commercial statistical package (Instat; Graph-pad, San Diego, CA).
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RESULTS |
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GIP receptors on ECV 304 cells and HUVEC have previously been identified (31). To determine whether GIP stimulates proliferation, we examined GIP effects on [3H]thymidine incorporation in HUVEC (Fig. 1A) and in ECV 304 (Fig. 1B). GIP concentrations as low as 10-13 M significantly stimulated [3H]thymidine incorporation in HUVEC with an EC50 of 1 pM. Increasing GIP concentrations to 0.01 nM and above had no further stimulatory effect. In fact, the GIP dose-response curve appeared to be bimodal, with supraphysiological doses of GIP (10 nM and above) still significantly elevated over control but below the peak response at 0.1 nM GIP.
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GIP also stimulated [3H]thymidine incorporation in ECV 304 cells, but the dose-response curve was shifted to the right with a calculated EC50 of 2.5 nM. A significant increase in [3H]thymidine incorporation was observed at 10 nM GIP, and increasing the agonist concentration provoked a corresponding increase in [3H]thymidine incorporation (Fig. 1B). The response was greatest at 1 µM GIP.
GIP Increases ET-1 Secretion in HUVEC but not in ECV 304
ET-1 is a known mitogen, and stimulated release from EC is regulated by a wide variety of factors, including glucose, cytokines, and mechanical factors (16). To determine whether GIP-induced elevations in [3H]thymidine incorporation were a direct or an indirect effect mediated by ET-1, we first measured GIP's ability to stimulate ET-1 secretion. HUVEC were stimulated with increasing concentrations of GIP (Fig. 2A), and GIP significantly increased ET-1 secretion at a concentration as low as 0.1 nM. Higher concentrations of GIP dose-dependently increased ET-1 secretion with an EC50 of 4.6 nM. In contrast, if ECV 304 cells were used, GIP had no significant effect on ET-1 secretion (Fig. 2B). To assess the underlying mechanism of this effect, we performed microarray analysis of GIP-stimulated HUVEC and ECV 304. HUVEC were stimulated for 6 h with GIP (0.1 nM), and 59 genes were upregulated, including a significant increase in endothelin-converting enzyme (Table 1). When the concentration of GIP was increased to 10 nM, 446 genes were now upregulated at least twofold, although the endothelin-converting enzyme was again upregulated (data not shown). In contrast, when ECV 304 cells were stimulated with 0.1 nM GIP, the expression of 66 genes was significantly changed. However, there was no change in the expression of endothelin-converting enzyme in ECV 304 in response to GIP (Table 2).
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BQ-788 Blocks GIP-Stimulated [3H]Thymidine Incorporation in HUVEC but not in ECV 304
To evaluate the role of ET-1 secretion from HUVEC, we utilized the ETBR blocker BQ-788. BQ-788 by itself, at a concentration of 10-6 M, had a small but statistically significant effect on increasing [3H]thymidine incorporation in HUVEC (Fig. 3). However, when increasing concentrations of BQ-788 were added to HUVEC stimulated with GIP (1 nM), [3H]thymidine incorporation was completely inhibited (Fig. 3). In ECV 304 cells, BQ-788 by itself (100 nM) also stimulated [3H]thymidine incorporation; however, when added together with GIP (100 nM)-stimulated cells, there was an additive (rather than inhibitory) effect on [3H]thymidine incorporation (Fig. 4).
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BQ-610 has no Effect on GIP-Stimulated [3H]Thymidine Incorporation
EC cells contain the ETBR predominantly, whereas smooth muscle cells mainly express the ETA receptor. To evaluate whether block of the ETA receptor had any effect on HUVEC proliferation, we used the selective ETA blocker BQ-610. BQ-610 at concentrations ranging between 10 nM and 1 µM had no effect on GIP-stimulated [3H]thymidine incorporation in HUVEC (Fig. 5), consistent with the low expression of the ETA receptor in EC.
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ET-1 and GIP have Additive Effects on Stimulated [3H]Thymidine Incorporation in ECV 304 but not in HUVEC
To further evaluate the role of ET-1 in GIP-stimulated [3H]thymidine incorporation, we examined the effect of combining ET-1 and GIP (Fig. 6A) in HUVEC. Both GIP (1 nM) and ET-1 (10 nM) alone significantly stimulated [3H]thymidine incorporation. However, when combined, there was no synergistic or additive effect on proliferation. In contrast, if ECV 304 were used (Fig. 6B), not only did GIP and ET-1 by themselves significantly stimulate [3H]thymidine incorporation, there was also an additive effect on proliferation by the combination of these two agonists.
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DISCUSSION |
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In a previous publication (31), we reported that the splice variants appeared to be differentially linked to different transduction pathways; the present data suggest, in addition, that the splice variants also have different functional links. As previously mentioned, the GIP receptor belongs to the G protein-coupled seven-transmembrane domain family of receptors, which are dually coupled to two signal transduction mechanisms: phosphoinositol-phospholipase C and adenylate cyclase. Additional diversity in the function of this family of receptors comes from the existence of both different receptor isoforms (e.g., VIP or PACAP) and splice variants [e.g., calcitonin (19), PACAP (24), CRF (6), and GHRH (27)]. Specifically, splice variants of the calcitonin receptor, which differ in the second extracellular domain, lead to differences in binding affinity for the hormone (11), whereas differences in the third intracellular loop of PACAP result in splice variants that display differences in their coupling to the signal transduction pathways (24). Thus there is precedence for the possibility that GIP receptor splice variants may be serving different functions in vascular endothelial cells.
Our data are consistent with the well-known mitogenic effect of ET-1 and
the preferential expression of ETB receptors in endothelial cells
(16,
18,
22). ET-1 is a potent
vasoconstrictor, and if one of the GIP receptor splice variants linked to ET-1
secretion were to be expressed in the hepatic artery, this would account for
the observed postprandial vasoconstriction. A difference in sensitivity to GIP
was also observed between HUVEC and ECV, with HUVEC responding to
concentrations of GIP as low as 10-13 M with an increase
in [3H]thymidine incorporation, whereas ECV required a
concentration of 10-8 M. Interestingly, the
dose-response curve for [3H]thymidine incorporation in response to
GIP was biphasic in HUVEC (Fig.
1A), with concentrations of GIP of 10 nM and above
actually leading to a decline in [3H]thymidine incorporation. This
biphasic response in [3H]thymidine incorporation was not observed
for ECV 304 (Fig. 1B).
We have previously demonstrated that GIP at 10 nM increased protein kinase A
activity in HUVEC but not in ECV 304 cells
(31). cAMP can either inhibit
or stimulate cell growth, depending on the cell type studied. cAMP effects on
the MAP kinase (or ERK kinase), cell cycle progression, or other
ERK-independent pathways have all been implicated in its inhibitory effect on
cell proliferation (25). In
endothelial cells, in particular, elevations in cAMP have been shown to
inhibit the mitogenic effects of vascular endothelial growth factor and bovine
fibroblast growth factor (7).
Thus it is possible that, in the case of HUVEC, GIP-induced elevations in cAMP
lead to an inhibition of proliferation that is not observed in ECV 304 because
GIP does not increase cAMP in these endothelial cells.
An unexplained finding is that the ETBR blocker BQ-788 had a small but significant effect on [3H]thymidine incorporation in HUVEC and ECV 304 (Figs. 3 and 4). BQ-788 may result in increased secretion of ET-1 from the endothelium, so this may be partially responsible for the observed small agonist effect of this compound (20). Nevertheless, BQ-788 is still effective in blocking GIP effects on [3H]thymidine incorporation in HUVEC but not in ECV 304.
In summary, GIP stimulates endothelial cell proliferation, and the mechanism for this effect is different in two closely related endothelial cell lines. Differences in GIP receptor splice variants between these two endothelial cell lines may account for the observed differences in response. GIP receptors are widely distributed in the body and can have different and sometimes opposing actions. We speculate that, in vivo, part of the differences in tissue responses to GIP may relate to both differences in GIP receptor splice variants and differences in the GIP concentration required to activate the tissue [related to differences in GIP receptor number (3)]. Further studies are currently in progress, characterizing the individual GIP receptor splice variants.
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DISCLOSURES |
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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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REFERENCES |
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