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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 1993-1999
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the New York University School of Medicine and Kaplan Cancer
Center, New York, NY.
Angiopoietin-1 (Ang-1) is required for developing vessels, and its
absence leads to defects in vessel remodeling. Ang-1 has been
identified as the ligand for the tyrosine kinase receptor Tie-2, which
is expressed specifically on endothelial cells and early hematopoietic
cells. In studying the role of Tie-2 and Ang-1 in megakaryocytopoiesis,
3 alternatively spliced species of Ang-1 mRNA (Ang-1.3 kb, Ang-0.9 kb,
and Ang-0.7 kb) were identified in addition to the full-length Ang-1
(Ang-1.5 kb), in the megakaryocyte cell line CHRF by reverse
transcription-polymerase chain reaction (RT-PCR), and then cloned and
sequenced. The expression of 3 alternatively spliced isoforms of Ang-1
was confirmed by RT-PCR using specific primer pairs derived from
junction sites and the 3' end of Ang-1 cDNA, and it was further
demonstrated by nuclease protection assay, Northern blotting, and
immunoblotting in CHRF cells. Expression of the Ang-1.3 kb isoform was
also detected in human primary fibroblast cell line FS4, breast cancer
cell line MDAMB-468, and CD34+CD41+ cells
of fetal liver and platelets. The function of the 1.5-kb, 1.3-kb, and
0.9-kb isoforms was examined. Recombinant proteins Ang-1.5 and 0.9 kb
bind strongly to the recombinant Tie-2 receptor (Tie-2-Fc), whereas the
1.3-kb isoform does not. The Ang-1.3 kb isoform binds to the 1.5-kb
isoform. Ang-1.5 kb, but not the 1.3-kb and 0.9-kb isoforms, induces
tyrosine phosphorylation of Tie-2 in human umbilical vein endothelial
cells. These data suggest that isoforms 1.3 kb and 0.9 kb could serve
as dominant negative molecules for the full-length Ang-1. The possible
involvement of the newly identified Ang-1 isoforms in angiogenesis and
in growth and differentiation of hematopoietic progenitor cells
provides a greater complexity to these processes.
(Blood. 2000;95:1993-1999)
Angiogenesis, the process of new growth of blood
vessels, is essential for supporting embryonic development, wound
healing, and tumor growth. It also plays a role in atherosclerosis,
diabetic retinopathy, and thrombosis. Various growth factors, including fibroblast growth factor (FGF) and vascular endothelial growth factor
(VEGF) have been implicated in the regulation of vessel formation.
Although FGF is a mitogen for a wide range of cell types, VEGF is a
mitogen for vascular endothelial cells,1,2 and it appears
to play an important role in regulating the function of endothelial
cells in normal and disease states.
Recently, angiopoietin-1 (Ang-1), a ligand for the Tie-2 receptor, was
isolated by secretion-trap expression cloning.3 Ang-1
signals through a tyrosine kinase receptor (Tie-2/Tek) that is
expressed specifically on endothelial cells and early hematopoietic cells.4-7 Mice engineered to lack Ang-1 or its receptor
express severe vascular abnormalities during
embryogenesis.8 Unlike VEGF, Ang-1 is incapable of
mediating endothelial cell growth or tubule formation. However, in the
absence of Ang-1, endothelial cells are poorly associated with the
underlying matrix and do not properly recruit and associate with
periendothelial supporting cells.3,8 Ang-1 has been shown
to be able to induce the migration and sprouting of endothelial
cells,9,10 and the overexpression of Ang-1 in transgenic
mice induces increased vascularization.11 Thus, Ang-1
appears to be involved in a later stage of vessel development.
Angiopoietin-2, a natural antagonist for the Tie-2 receptor that
disrupts in vivo angiogenesis, has recently been described.12 Both Ang-1 and Ang-2 could modulate
VEGF-induced postnatal neovascularization.13,14 Thus it is
likely that angiogenic processes depend on the sequential and
cooperative effects of the members of VEGF and angiopoietin families.
The endothelial and hematopoietic lineages are closely linked. Several
receptor tyrosine kinase molecules are expressed on both lineage cells,
including Flk-1, Flt-1, Tie-1, and Tie-2. Tie receptor kinases are of
special interest because they are expressed in very early stages of
hematopoietic and endothelial cell development.4-7 Indeed,
Tie-1 was first isolated from a human chronic myeloid leukemia cell
line K562, and its expression was up-regulated during in vitro
megakaryoblastoid differentiation of certain leukemia cell
lines.15-17 Tie-1 expression was also found in vivo in
megakaryocytes, hematopoietic stem cells, and some B cells of adult
bone marrow or umbilical cord blood cells.18,19 Tie-2 is
also expressed in bone marrow hematopoietic stem cells in a subset of
CD34+ or c-KIT+ cells.20
In studying the role of Tie-2 and its ligand, Ang-1, in
megakaryocytopoiesis, we discovered the presence of 4 Ang-1 isoforms in
the megakaryocyte cell line, CHRF. In this article we report on their
cloning, sequencing, and function (ability to bind and phosphorylate
Tie-2), and we provide suggestive evidence that 2 of these isoforms may
act as dominant-negative inhibitors.
Cell culture
Reverse transcription-polymerase chain reaction
Primers for reverse transcription-polymerase chain reaction A series of RT-PCR experiments was conducted using pairs of oligonucleotide primers corresponding to different regions and isoforms of the Ang-1 sequences. The detection of Ang-1 expression was performed using sense primer 5'-GGAAGTCTAGATTTCCAAAGAGGC-3' and antisense primer 5'-CTTTATCCCATTCAGTTTTCCATG-3', corresponding to sequences 1306-1326 bp and 1711-1734 bp, respectively, designed to give a 429-bp product numbered according to the Ang-1 cDNA sequence submitted to GenBank, accession number U83 508. Amplification of the full-length coding region of Ang-1 was generated with sense primer 5'-GCTGGCAGTACAATGACAGGT-3' (identical to 5' end) and antisense primer 5'-TCAAAAATCTAAAGGTCGAAT-3' (complementary to 3' end). Two sense primers designed to be located within the junction sites of alternatively spliced mRNA were 5'-GGAATATAAAATGGTTGTATTTAA-3' (specific for Ang-1.3 kb), and 5'-GTGGCTGCAAAAAGTGTTTTGC-3' (specific for Ang-0.9 kb). The sense primer specific for Ang-1.3 kb was paired with an antisense primer complementary to the 3' end of Ang-1 cDNA to give a 312-bp product (for detection of the 1.3-kb isoform). The sense primer specific for Ang-0.9 kb was paired with an antisense primer 5'-ATCGCTTCTGACATTGCGCTT-3' (1807-1827 bp of the Ang-1 sequence) to give an amplification fragment of 595 bp. Primers used for Ang-2 were 5'-GGCAGCGTTGATTTTCAGAGGACT-3' and 5'-TTTAATGCCGTTGAACTTATTTGT-3', corresponding to 1340-1363 bp and 1746-1769 bp of the published sequence.Cloning and sequencing analysis cDNA for individual Ang-1 mRNA species was obtained by RT-PCR using 2 primers derived from either the 5' or the 3' end of the translated sequences of human Ang-1. The amplified product was fractionated on an agarose gel. Several DNA fragments, ranging in size from 0.7 to 1.5 kb, were observed. These RT-PCR products were cloned into a pGEM-T Easy vector (Promega, Madison, WI). The cloned cDNA was then sequenced with T7 and SP6 sequencing primers and with Ang-1-specific primers. Sequencing data were compared with the published Ang-1 sequence. Isoform sizes were calculated from the sequence data. In some experiments, specific bands from RT-PCR products were excised. The DNA was then eluted, cloned, and sequenced.Northern blot analysis The polyadenylated mRNA fraction was isolated by binding to oligo(dT) cellulose, fractionated by electrophoresis on 1% agarose gel in 6.7% formaldehyde, and transferred to a Genescreen Plus nylon membrane (NEN Life Science Products, Boston, MA). The membrane was hybridized at 42°C overnight with a 32P-dCTP-labeled probe either the 1.5-kb full-length Ang-1, an approximately 0.5-kb
alternatively spliced fragment of Ang-1 absent in the 0.9-kb and 0.7-kb
isoforms, or a 0.17-kb alternatively spliced fragment of Ang-1 absent
in the 1.3-kb and 0.7-kb isoforms. Blots were sequentially washed with
varying dilutions of SSC, the last 0.1 × SSC at 65°C for 30 minutes. Autoradiography was carried out at  70°C with
intensifying screen.
Nuclease protection assays cDNA fragments derived from alternatively spliced sites of the 1.3- or 0.9-kb isoform were cloned into pGEM-T vector (Promega) to generate probes 1 and 2. Antisense probes were made using T7 polymerase and -32p-UTP. Total RNA (20 µg) extracted from CHRF cells or DU145
cells was used for solution hybridization according to the
manufacturer's protocol (Ambion, Austin, TX). Addition of
hybridization probe 1 to detect the 1.3-kb isoform, followed by RNase
digestion, gave a protected band of 262 bp, whereas addition of
hybridization probe 1 to detect isoforms 1.5 or 0.9 kb, followed by
RNase digestion, gave bands of 219 and 43 bp. Hybridization probe 2 protected the 0.9-kb isoform from RNase digestion, resulting in a
249-bp band, and protected the 1.5-kb and 1.3-kb isoforms from RNase
digestion, resulting in 146-bp and 103-bp bands. See "Results"
for detailed strategy.
Immunoblotting Cells were washed twice with phosphate-buffered saline and lysed in lysis buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris, pH 7.4, 50 µg/mL pepstatin, 20 µg/mL aprotinin, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L EDTA, 0.2 mmol/L sodium orthovanadate, and 0.5% NP-40). One hundred micrograms protein, determined by Bio-Rad protein assay, was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a membrane, incubated with blocking solution (2% powdered milk, 0.2% Tween 20 in phosphate-buffered saline), and reacted with a specific rabbit anti-Ang-1 antibody, kindly provided by Dr George Yancopoulos (Regeneron Pharmaceuticals, NY) or with a goat anti-Ang-1 antibody specific for the N-terminal end (reactive with anti-1.5 kb and anti-1.3 kb isoforms) or C-terminal end (reactive with 1.5-kb isoform but not 1.3-kb isoform) supplied by Santa Cruz Biotechnology (Santa Cruz, CA). After washing, a peroxidase-conjugated second antibody was applied and chemiluminescence generated by incubation with ECL reagents (Amersham Life Science).Protein isoform expression and purification cDNA corresponding to the full-length Ang-1 and various isoforms (without signal peptide) was cloned in frame into the pGE × 4T1 vector (Pharmacia) and then transformed to BL21 Escherichia coli. Recombinant GST-Ang-1 fusion proteins were purified from bacterial lysates by affinity chromatography on a glutathione Sepharose 4B column (Pharmacia).Pull-down assay for isoform-isoform binding Two different methods were used. In the first method, 35S-radiolabeled Ang-1 isoforms 1.3 kb, 0.9 kb, and control protein luciferase were prepared by transcription/translation in vitro and incubated with the GST-Ang-1.5-kb fusion protein in binding buffer (10 mmol/L Tris, pH 7.6, 100 mmol/L NaCl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1% nonfat dry milk, 5 mmol/L MgCl2, 0.05% NP40, and 8% glycerol) at 4°C for 60 minutes. The materials were then incubated with glutathione Sepharose 4B beads for 30 minutes and were shaken at 4°C. After washing with binding buffer, the proteins bound to the beads were solubilized in sample lysis buffer, resolved on 12% SDS-PAGE, and exposed to x-ray film. In the second method, COS cells were transfected with a mixture of 1.5-kb and 1.3-kb isoforms or the 1.5-kb isoform alone. Cells were lysed with sample lysis buffer, and isoforms were precipitated with an anti-Ang-1 antibody reactive with the 1.5-kb isoform, not the 1.3-kb isoform (C-terminal antibody) plus or minus protein G beads. Samples were then analyzed by Western blot with an anti-Ang-1 antibody reactive with both the 1.5- and 1.3-kb isoforms (N-terminal antibody). The specificity of the antibodies was verified independently by Western blot against the specific isoforms.Ligand-receptor binding assay Binding of the Ang-1 isoforms to the Tie-2 receptor was also evaluated by ligand-receptor precipitation in the presence of 5% bovine serum albumin. In vitro ribosome-translated 35S-labeled Ang-1 isoforms were incubated with recombinant Tie-2-Fc protein, followed by precipitation with protein G beads. After washing, the proteins bound to the resin were solubilized in sample lysis buffer and resolved on 12% SDS-PAGE followed by autoradiography.Tyrosine phosphorylation assay Human umbilical vein endothelial cells (HUVEC) were plated in T-75 flasks, cultured to confluence, and serum starved for 4 hours. Conditioned media, collected from COS cells transiently transfected with various plasmids of Ang-1 isoforms, were placed on HUVEC for 10 minutes. Cells were then solubilized with lysis buffer (1% Triton X-100, 10 mmol/L sodium pyrophosphate, 100 mmol/L sodium fluoride, 4 mmol/L EDTA, 2 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 100 mmol/L leupeptin, and 50 mg/mL aprotinin). Tie-2 protein was immunoprecipitated from the cell lysates with a goat anti-Tie-2 antibody (1 µg/mL) and autophosphorylation of tyrosine residues was evaluated with antiphosphotyrosine mAb 4G10 (Upstate Biotechnology, NY) by Western blotting, followed by chemiluminescence using ECL (Amersham).
Detection of Ang-1 expression in human cells by RT-PCR The expression of Ang-1 mRNA was analyzed in several tumor cell lines and normal tissues. Positive signals were obtained from RNA of CHRF, MDA-MB468, and MDA-MB231 human tumor cell lines and from human FS4 fibroblasts, fetal liver CD34+CD41+ cells, and cord blood CD34+ cells and platelets, whereas none were obtained from the RNA of MCF7 and DU145 human tumor cell lines and several normal human tissue specimens (Table 1, Figure 1). Ang-2 was not expressed in the cell lines studied (data not shown).
Alternatively spliced species of Ang-1 mRNA identified by RT-PCR, RNase protection, Northern blot analysis, and immunoblot Using RT-PCR to analyze the expression of Ang-1 mRNA in CHRF cells, we noted an extra band migrating at approximately 262 bp with ethidium bromide staining, in addition to the 429 bp-band predicted because of the pair of primers used (Figure 2). Another extra band was observed in FS4 cells and MDA-MB468 cells (see below). The extra band was excised from the gel, and the DNA was eluted, cloned, and sequenced. Sequencing indicated that the 262-bp band represented a fragment of an alternatively spliced form of Ang-1 mRNA, later identified as the 1.3-kb isoform. To examine further the expression of multiple isoforms of the Ang-1 coding region in human cells, a pair of primers designed to span the coding region of the Ang-1 gene was applied for RT-PCR analysis using RNA extracted from CHRF cells. Several amplified products were detected. After examining 50 clones and selecting 8 for sequencing, 3 alternative transcripts (Ang-1.3 kb, Ang-0.9 kb, and Ang-0.7 kb) were identified, as was the published Ang-1.5 kb sequence for the coding region (Figures 3 and 4). To confirm the presence of these transcripts, new sets of primers were designed with the 5' end straddling the apparent splice junction site. A set of primers, used to detect the 1.3-kb isoform and designed to give a 312-bp product, is shown in Figure 5A. This isoform was found in FS4, MDA-MB468, and CHRF cells, not in DU145, MDA-MB231, and MCF7 cells (Table 1). The primer pair used to detect the 0.9-kb isoform and designed to give a 595-bp product is shown in CHRF cells (Figure 5B). Also shown is a 428-bp product that represents a double alternatively spliced isoform, Ang-0.7 kb. Neither of these isoforms was found in MDA-MB468, MDA-MB231, or FS4 cells (data not shown).
Ang-1.5 kb and Ang-0.9 kb isoforms bind to the Tie-2 receptor
Ang-1.3 kb isoform, but not 0.9 kb-isoform, binds to the 1.5-kb
isoform
Ang-1.5 kb, but not other isoforms, induces tyrosine phosphorylation
of Tie-2
These data indicate the presence of Ang-1 in 4 of 6 human cell lines
examined (MDA-MB468 and MDA-MB231 breast, CHRF megakaryocyte, and FS4
fibroblast), fetal liver CD34+CD41+ cells, cord
blood CD34+ cells and platelets with alternatively spliced
mRNA species in 3 of the cell lines (CHRF, MDA-MB468, and FS4), and
fetal liver CD34+CD41+ cells and platelets.
Submitted May 20, 1999; accepted July 17, 1999.
Supported by National Institutes of Health grant HL-13336-26 and by
grants from the Helen Polonsky Research Fund and the Dorothy and
Seymour Weinstein Research Fund.
Reprints: Yao-Qi Huang, New York University School of Medicine
and Kaplan Cancer Center, 550 First Avenue, New York, NY 10016;
e-mail: huangy02{at}mcrcr6.med.nyu.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction
-actin was used as an
internal control (lower panel).
