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INTRODUCTION |
Vascular endothelial growth factor
(VEGF),1 also known as
vascular permeability factor, is a potent angiogenic and endothelial cell-specific mitogen (1, 2). VEGF is expressed and secreted at low
levels by most normal cells but constitutively expressed at high levels
by many human tumors and tumor cell lines (1-4). Hypoxia up-regulates
VEGF expression and several studies have demonstrated that increase in
transcription alone does not account for all of the increase in VEGF
mRNA (3-8). The post-transcriptional regulation of VEGF mRNA
stability also plays a critical role in the observed hypoxic induction
(6-8).
Post-transcriptional regulatory mechanisms, especially modulation of
mRNA stability, has been shown to play a major role in gene
expression (9). The turnover rate of a given mRNA can be determined
by interactions of trans-acting factors with specific cis-element
located within 3'-untranslated regions (3'-UTR) (9, 10). Many labile
mRNAs, including those that encode lymphokines, cytokines,
transcription factors, and proto-oncogenes, contain AU-rich elements
(AREs) in their 3'-UTR (9). Identification of the interaction of AREs
with trans-acting proteins has been the first step in understanding the
molecular regulation of mRNA stability (9, 10). The presence of a
reiterated pentamer (AUUUA)n in many AREs has been shown to be
associated with rapid mRNA turnover and translation attenuation
(10-12). In the case of granulocyte-microphage colony-stimulating
factor, c-Fos, and c-Myc mRNAs, deletion of the ARE region enhances
their stability, and insertion of the region into the 3'-UTR of a
normally stable globin mRNA significantly destabilizes it (10-15).
A variety of AUUUA-binding proteins have been identified, and examples
of these include (i) a 32-kDa nuclear protein from HeLa cells (15); (ii) AU-A, AU-B, and AU-C, 30-43-kDa nuclear and/or cytoplamic proteins from human T lymphocytes (16, 17); (iii) AUBF, a heterotrimeric protein formed by 15-, 17-, and 19-kDa subunits and
present in both nucleus and cytoplasm (18, 19); and (iv) AUBP, a 36-kDa
cytoplasmic protein from human spleen identified as
glyceraldehyde-3-phosphate dehydrogenase (20). However, the mechanisms
of how these proteins affect mRNA turnover remains unclear.
Our previous work identified a 126-base hypoxia stability region (HSR)
in human VEGF 3'-UTR that is critical for the stabilization of VEGF
mRNA under hypoxia (21). This region is able to form seven
hypoxia-inducible mRNA-protein complexes (21). Here we report hnRNP
L as a protein that interacts with the VEGF HSR and forms a
hypoxia-inducible 60-kDa RNA-hnRNP L complex. The cytoplasmic hnRNP L
specifically interacted with VEGF mRNA in hypoxic cells in
vivo and regulated VEGF mRNA stability. Thus, we propose that a specific interaction of hnRNP L with VEGF mRNA may play an
important role in hypoxia-induced post-transcriptional regulation of
human VEGF mRNA stability.
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MATERIALS AND METHODS |
Antibodies and Oligodeoxyribonucleotides--
Monoclonal
antibodies 4D11 (anti-hnRNP L) and 4F4 (anti-hnRNP C) were generously
provided by Dr. Gideon Dreyfuss (University Pennsylvania, Philadelphia)
(22, 23). All of the oligodeoxyribonucleotides used in the study were
synthesized from Genemed Synthesis (San Francisco, CA).
Cell Lines and Culture Conditions--
Human melanoma cell line
M21 was obtained from Dr. Romaine Saxton (UCLA, Los Angeles, CA). Cells
were routinely grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 10 units/ml penicillin, and 10 µg/ml
streptomycin. Cells were cultured under either normoxic conditions (5%
CO2, 21% O2, 74% N2) in a
humidified Queue incubator (Asheville, NC) at 37 °C or hypoxic
conditions (5% CO2, 3% O2, 92%
N2) in a humidified triple gas Heraeus incubator (model
6060, Hanau, Germany) at 37 °C.
Preparation of Cytoplasmic and Nuclear Extracts--
Cytoplasmic
and nuclear extracts were obtained as described by Claffey et
al. (21). Following exposure to normoxia or hypoxia (3%
O2), M21 cells were washed three times in ice-cold PBS
followed by lysis in 1% Triton X-100 lysis buffer containing 50 mM Hepes, pH 7.5, 10 mM sodium pyrophosphate,
150 mM NaCl, 100 mM NaF, 0.2 mM
NaOVa4, 1 mM EGTA, 1.5 mM
MgCl2, 10% glycerol, and 5 mM
4-(2-aminoethyl)benzene-sulfonyl fluoride (Sigma). The cytoplasmic
extract was collected and centrifuged at 14,000 × g
for 15 min, and the nuclei pellet were further extracted with the 1%
Triton X-100 lysis buffer containing 400 mM NaCl for 15 min
on ice. The nuclear extract was recovered after centrifuged at
14,000 × g for 15 min.
In Vitro Transcription--
The sense and antisense HSR of human
VEGF were transcribed in vitro as described previously (21),
using T7 RNA polymerase transcription of NotI-linearized and
T3 RNA polymerase transcription of EcoRI-linearized plasmid,
respectively. Both biotin-UTP-labeled and
[32P]UTP-labeled RNA transcripts were generated using an
RNA in vitro transcription kit (Stratagene, La Jolla, CA)
according to the manufacturer's protocol and then treated with
RNase-free DNase (Promega, Madison, WI) for 15 min at 37 °C. The
[32P]UTP-labeled transcripts were extracted once with
phenol:chloroform and loaded onto RNase-free G-50 spin columns
(Boehringer Mannheim) to remove free ribonucleotides. The
biotin-UTP-labeled sense and antisense HSR transcripts were
precipitated with ethanol and dissolved in diethyl
pyrocarbonate-treated H2O. The transcripts were then immobilized to an avidin-Sepharose 4B column (Amersham Pharmacia Biotech, Uppsala, Sweden) at 4 °C for 1 h and used in
mRNA-binding protein affinity purification.
RNA Binding and UV Cross-linking (RNA-UVXL)--
RNA-UVXL was
performed as described earlier (21). Radiolabeled RNA transcripts
(250,000 cpm/reaction) were incubated with cytoplasmic or nuclear
proteins (40 µg/reaction) in 30 µl of RNA binding buffer containing
10 mM Hepes, pH 7.5, 5 mM MgCl2, 50 mM KCl, 0.5 mM EGTA, 0.5 mM
dithiothrietol, 10% glycerol, 100 µg/ml tRNA, and 5 mg/ml heparin
for 20 min at 30 °C. The mixtures were UV cross-linked by UV
Stratalinker (Stratagene) at room temperature for 10 min (total energy
1800 J/cm2) followed by RNase digestion (40 units of RNase T1
(Boehringer Mannheim) and 1 µg of RNase A (Sigma)) for 15 min at room
temperature. The sample was then denatured in SDS-PAGE sample buffer
under reducing conditions and RNA-protein complexes were analyzed by
8% SDS-PAGE and autoradiographed with Eastman Kodak Co. MR film.
Affinity Purification of mRNA-binding
Proteins--
Cytoplasmic extracts were prepared after M21 cells were
cultured in hypoxia for 24 h. The lysate was sequentially loaded
onto poly(A)-, poly(U)-, antisense HSR- and sense HSR-Sepharose 4B RNA
affinity columns at room temperature. The sense HSR-Sepharose column
was then extensively washed with phosphate-buffered saline, 0.2%
Triton X-100. The mRNA-binding proteins were then eluted with 0.9 M NaCl and precipitated with 3 volumes of ethanol.
Immunoprecipitation--
M21 cytoplasmic lysates were incubated
with a 1:500 dilution of anti-hnRNP C ascites (4F4), anti-hnRNP L
ascites (4D11; both 4F4 and 4D11 were obtained from Dr. Gideon
Dreyfuss, University of Pennsylvania, Philadelphia, PA), or control
antibody (normal mouse serum) for 2 h at 4 °C. The lysates were
further incubated with 30 µl of protein A-Sepharose beads (Amersham
Pharmacia Biotech) for 2 h at 4 °C. Beads were recovered by
brief centrifugation, washed three times in lysis buffer, and RNA was
extracted for RT-PCR or denatured directly with SDS-PAGE buffer for
Western blot.
Western Blot--
The immunoprecipitated material was denatured
under reducing conditions, and proteins were analyzed by 8% SDS-PAGE.
Western blots were performed using a 1:5000 dilution of anti-hnRNP L
monoclonal antibody, anti-hnRNP C monoclonal antibody, and normal mouse
serum, followed by a 1:10,000 dilution of horseradish
peroxidase-conjugated goat anti-mouse IgG (Amersham Pharmacia Biotech)
in a blocking buffer containing 1% bovine serum albumin and 0.1%
Tween 20 in Tris-buffered saline. The blots were then developed with
the ECL system (Amersham Pharmacia Biotech).
RT-PCR and Southern Blot Detection--
The protein
A-Sepharose-bound RNA was extracted with an equal volume of Trizol
(Life Technologies, Inc.) and
volume of chloroform. The
supernatant was collected after centrifugation at 14,000 × g for 15 min, and RNA was precipitated with an equal volume
of isopropyl alcohol at
80 °C for 1 h. Reverse transcription was carried out with oligo(dT) (15-mer) as primer and incubated at
42 °C for 1 h in the presence of reverse transcriptase and dNTPs (Boehringer Mannheim). The transcribed cDNA was collected after removal of free dNTPs through RNase-free G-50 spin column (Boehringer Mannheim). PCR amplification was carried out with a
Taq DNA polymerase and two VEGF primers (VEGF 5' primer:
5'-TATGCGGATCAAACCTCAC-3', corresponding to nucleotides 374-393 of
VEGF coding region encoding amino acids 82-88 of the mature protein;
VEGF 3' primer: 5'-ATAACATTAGCACTGTTAATTT-3', corresponding to
nucleotides 435-457 3' to the translation strip codon,
GenBankTM accession number AF022375) and amplified for 30 cycles. The 716-base-long PCR products were resolved by 1% agarose gel
electrophoresis and detected by Southern blot with a
[32P]dCTP-labeled human VEGF165
AccI/NcoI fragment (823 base pairs) encompassing
the coding region and 330 base pairs of 3'-UTR of the full-length
cDNA. Probes were prepared by the random-primed synthesis method
using the Multiprime kit (Amersham Pharmacia Biotech). DNA was blotted
by capillary transfer to a nylon membrane (NEN Life Science Products)
in 10× SSC. Blots were cross-linked with UV Stratalinker 1800 (Stratagene), baked at 80 °C for 15 min, and prehybridized for 2-4
h at 65 °C. Hybridization was carried out overnight at 65 °C, and
blots were washed at 0.5% SDS, 0.1% SSC at 55 °C before they were
exposed to Kodak MR film.
Oligonucleotide Transfection and Northern Blot
Detection--
Antisense oligodeoxyribonucleotides, AS1 (1 µM) and AS3 (1 µM), were transfected into
M21 cells by Lipofectin following the instructions of the supplier
(Life Technologies, Inc.). The transfected cells were then cultured in
hypoxia for 8 h, and total cellular RNA was isolated by using the
RNeasy RNA extraction kit (Qiagen, Chatsworth, CA) according to the
manufacturer's instructions. For the VEGF mRNA stability study, 5 µg/ml actinomycin D was added to AS1, AS3, or mock-transfected M21
cells after 6 h of hypoxia incubation. The time intervals of
actinomycin D treatment were 30 min, 1 h, and 2 h, and the
total hypoxia incubation time was 8 h. Total RNA was isolated and
electrophoresed in 1% agarose gels containing 2.2 M
formaldehyde (10 µg/lane). RNA was blotted by capillary transfer to a
nylon membrane (NEN Life Science Products) in 10× SSC. Blots were
cross-linked with UV Stratalinker (Stratagene), baked at 80 °C for
15 min, and prehybridized for 2-4 h at 65 °C. Hybridization was
carried out overnight at 65 °C with a
[32P]dCTP-labeled human VEGF165
AccI/NcoI fragment and GLUT-1 BamHI fragment (2.47 kilobase pairs). A ribosome-associated protein cDNA,
36B4, was used as a control (21). Blots were washed with 1% SDS, 1%
SSC at 55 °C and exposed to Kodak MR film. VEGF was normalized to
36B4 expression using PhosphorImager analysis (Molecular Dynamics,
Sunnyvale, CA).
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RESULTS |
Indentification of VEGF 3'-HSR mRNA-binding Proteins--
The
post-transcriptional regulation of VEGF mRNA stability in response
to hypoxia may, in part, be due to the interaction of VEGF 3'-UTR with
specific binding protein(s) (6-8, 27, 28). Our previous work
identified a 126-base HSR in human VEGF 3'-UTR (Fig.
1A), which is critical for
hypoxia-induced human VEGF mRNA stability (21). The HSR formed
seven RNA-protein complexes with M21 human melanoma cell cytoplasmic
proteins with apparent molecular masses of 90, 88, 72, 60, 56, 46, and
40 kDa in RNA-UVXL (21).

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Fig. 1.
VEGF HSR-binding proteins in cytoplasm and
nucleus. Panel A, the human VEGF 3'-HSR (3'-UTR
332-457) sequence. The single AUUUA pentamer is underlined.
Panel B, cytoplasmic (c) and nuclear
(n) extracts were prepared from M21 cells cultured in
normoxia (N) or 24-h hypoxia (H). The VEGF HSR
RNA-protein complexes were UV-cross-linked and separated by 8%
SDS-PAGE under reducing conditions. The apparent molecular masses of
each RNA-protein complex are indicated by arrows. Background
binding in the absence of extract is indicated as B.
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Studies have shown that RNA-binding proteins are also present in
nuclear compartments (15-17). Thus, we prepared both cytoplasmic and
nuclear extracts from hypoxic or normoxic cultured M21 cells and
compared RNA-protein complex formation by RNA-UVXL with
32P-labeled VEGF HSR. As shown in Fig. 1B, 24-h
hypoxia (3% O2) substantially up-regulated the formation
of the 90-, 88-, 72-, and 60-kDa complexes when compared with
cytoplasmic extracts made from normoxic (21% O2) cells.
The 90-, 88-, 72-, and 56-kDa complexes were selectively cytoplasmic
with little or no nuclear localization (Fig. 1B).
Significantly higher levels of the 60-kDa complex were formed with
nuclear extracts than cytoplasmic extracts, and the complex was
markedly increased by hypoxia treatment in both extracts (Fig.
1B). Other than the 60-kDa complex, the nuclear extract also
formed three unique complexes with apparent molecular masses of 45, 48, and 120 kDa (Fig. 1B). Taken together, these results suggest
that proteins that form the 90-, 80-, 72-, and 56-kDa complexes with
HSR mainly appear in cytoplasm. In contrast, the protein that forms the
60-kDa complex with HSR is present in both nuclear and cytoplasmic
compartments with the majority in the nucleus. Thus, given the
distribution and the hypoxia induction of the 60-kDa complex, we
established a protocol to purify the RNA-binding protein in the 60-kDa complex.
Purification of the RNA-binding Protein in the 60-kDa RNA-Protein
Complex--
The VEGF HSR is a highly AU-rich element containing 54 A
bases and 52 U bases out of 126 bases, resulting in 43% A and 41% U
distribution (Fig. 1A). To purify the binding protein that
forms the 60-kDa complex, hypoxia-treated M21 cell lysates were loaded sequentially onto poly(A)-, poly(U)-, and antisense HSR-coupled columns
to remove nonspecific RNA-protein interactions prior to loading to the
sense HSR-coupled column. To ensure that the protein that formed the
60-kDa complex was not removed by poly(A)-, poly(U)-, and antisense
HSR-coupled columns, the flow-through from each column was collected,
and RNA-UVXL was performed with 32P-labeled HSR. As shown
in Fig. 2A, the poly(A) column
effectively interacted with proteins forming the 90- and 72-kDa
complexes and removed them from the cell lysates, whereas the poly(U)
column removed the proteins that formed the 88-, 56-, 46-, and 40-kDa complexes. Further incubation of the lysate with the antisense HSR
column completely removed proteins forming the 72-kDa complex, whereas
the 60-kDa complex selectively bound to the sense HSR column (Fig.
2A). These results suggest that the proteins forming the 90- and 72-kDa complexes can interact with poly(A) sequences, whereas
proteins forming the 88-, 56-, 46-, and 40-kDa complexes can interact
with poly(U) sequences. In contrast, the proteins present in the 60-kDa
complex only specifically interact with a unique sequence in the
HSR.

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Fig. 2.
Purification of RNA-binding protein that
forms the 60-kDa HSR-protein complex. Panel A, M21
cytoplasmic lysates (24-h hypoxia) were sequentially loaded onto four
different RNA-coupled Sepharose 4B columns: poly(A) (A),
poly-U (U), antisense HSR (AS), and sense HSR
(S). Flow-through from each column was collected and used
for RNA-UVXL analysis. The VEGF HSR RNA-protein complexes were analyzed
by 8% SDS-PAGE. Background binding in the absence of cytoplasmic
extract is indicated as B. Panel B, bound
materials on each of the four columns were denatured with reducing
SDS-PAGE sample buffer, separated with 6% SDS-PAGE, and visualized
with silver stain. The 60-kDa sense HSR-binding protein was eluted with
0.9 M NaCl from the sense HSR column.
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To analyze proteins that bound to the four mRNA columns, small
aliquots of the column material were directly analyzed in reducing 6%
SDS-PAGE. As shown in Fig. 2B, all four mRNA columns
interacted with numerous proteins. However, the sense HSR column
contained one unique protein band with an apparent molecular mass near
60 kDa (indicated by an arrow). When eluted with various
concentrations of NaCl, the 60-kDa protein was successfully released
from the sense HSR column with 0.9 M NaCl (Fig.
2B, indicated by an arrow). The purified 60-kDa
protein was then separated in a preparative 6% SDS-PAGE, stained with
Coomassie Blue, and sequenced after trypsin digest and HPLC
purification (Harvard Microchemistry Protein Sequencing Facility). A
16-amino acid sequence of an HPLC-purified peptide was determined as
SDALETLGFLNHYQMK, which is identical to hnRNP L, amino acid residues
522-537 (22).
The RNA-binding Protein in the 60-kDa Complex Is hnRNP L--
To
further confirm the RNA-binding protein in the 60-kDa complex as hnRNP
L, we used anti-hnRNP L monoclonal antibody to immunoprecipitate hnRNP
L from M21 cell lysates followed by detection of the 60-kDa complex
formation by RNA-UVXL. For hnRNP control, anti-hnRNP C monoclonal
antibody was used. Direct Western blot analysis of M21 cytoplasmic
extracts showed that the anti-hnRNP L monoclonal antibody identified
three proteins with apparent molecular masses of 66, 60, and 56 kDa,
with the 60-kDa band being the most abundant of the three (Fig.
3A), indicating that hnRNP L
is expressed as three different isoforms or differentially modified
proteins in M21 cells. Anti-hnRNP L immunoprecipitation removed all of
the 56- and 66-kDa bands and most of the 60-kDa hnRNP L band (Fig. 3A). Control antibodies (anti-hnRNP C and normal mouse
serum) did not interact with any of the three hnRNP L proteins (Fig. 3A). Fig. 3B shows that removal of hnRNP L
molecules by immunoprecipitation abolished formation of the 60-kDa
RNA-protein complex in RNA-UVXL, whereas formation of the other
RNA-protein complexes was unaffected. Immunoprecipitation with
anti-hnRNP C did not affect any of the seven HSR-protein complexes,
suggesting that hnRNP C is not one of the RNA-binding proteins present
in these complexes. Taken together, these results further prove that
hnRNP L is indeed the RNA-binding protein responsible for the 60-kDa
RNA-protein complex. Removal of hnRNP L from cell lysates did not
affect the formation of the other six RNA-protein complexes, suggesting
that hnRNP L does not form specific complexes with other RNA-binding
proteins interacting with VEGF HSR.

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Fig. 3.
hnRNP L protein is the trans-acting factor
that forms the 60-kDa protein HSR complex. M21
cytoplasmic extracts (24-h hypoxia) were immunoprecipitated with
anti-hnRNP L monoclonal antibody (L), anti-hnRNP C
monoclonal antibody (C), or normal mouse serum ( ). The
unbound proteins after immunoprecipitation were either analyzed by
Western blot with anti-hnRNP L monoclonal antibody (A) or
RNA-UVXL with 32P-labeled VEGF HSR (B). The
samples from both experiments were separated with 8% SDS-PAGE.
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Hypoxia Regulation of hnRNP Expression--
Hypoxia substantially
increases the steady state level of VEGF mRNA (4-8). The formation
of hnRNP L-VEGF 3'-HSR complex markedly increased when
cytoplasmic protein extracts were obtained from M21 cells exposed to
hypoxia (3% O2) as compared with those exposed to normoxia
(21% O2) (Fig. 1B). Thus, it was of interest to
compare the effect of hypoxia on synthesis and distribution of hnRNP L in nuclear versus cytoplasmic compartments. As shown in Fig.
4, M21 cells grown in normoxic conditions
contained high levels of hnRNP L, especially in the nuclear
compartment. A minor induction of hnRNP L can be seen under hypoxia in
both cytoplasm and nucleus, with the 56-kDa immunoreactive band in
cytoplasm being the most significantly increased (Fig. 4). Conversely,
the hnRNP C level in cytoplasm decreased to an undetectable range after
a 24-h hypoxia incubation, although it remained high in the nucleus
(Fig. 4). These results suggest that hypoxia differentially regulates
the distribution of hnRNPs in different cellular compartments.

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Fig. 4.
Regulation of hnRNP L and hnRNP C expression
by hypoxia. Cytoplasmic (c) and nuclear (n)
extracts were prepared from M21 cells after culture in normoxia
(N) or 24-h hypoxia (H). Proteins were separated
by 8% SDS-PAGE, and Western blot was performed with anti-hnRNP L
(L) or anti-hnRNP C (C) monoclonal antibodies.
Arrows indicate hnRNP L and hnRNP C proteins.
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Identification of hnRNP L mRNA Binding Site--
To define the
hnRNP L mRNA binding site, six antisense oligodeoxyribonucleotides
were synthesized from different regions of the HSR (Fig.
5A, AS1-AS6). To ensure the
stability of oligonucleotides in the RNA-UVXL, we synthesized
oligodeoxyribonucleotides instead of oligoribonucleotides. As shown in
Fig. 5B, AS1 (1 µM) efficiently blocked the
interaction of hnRNP L with 32P-labeled HSR and abolished
formation of the 60-kDa RNA·hnRNP L complex. The blocking of
HSR·hnRNP L complex formation can be seen as low as 0.05 µM AS1 (data not shown). AS6 abolished the formation of
the 90- and 88-kDa complexes and partially inhibited the HSR·hnRNP L
complex, whereas AS2, AS3, AS4, and AS5 did not show any effect (Fig.
5B). These results suggest that the complementary region of
AS1 on HSR contains the hnRNP L binding site and that hnRNP L interacts
with the single-stranded mRNA region. AS1 and AS6 lack sequence
homology; thus, the partial inhibition of hnRNP L interaction with HSR
observed with AS6 may be functioning by blocking the 90- and 88-kDa
complexes that may promote hnRNP L binding in some manner.

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Fig. 5.
Inhibition of the 60-kDa HSR·hnRNP L
complex formation with antisense oligodeoxyribonucleotides.
A, VEGF HSR sequence and the location of six chemically
synthesized antisense (AS) oligodeoxyribonucleotides.
B, blocking of HSR and hnRNP L interaction by AS
oligodeoxyribonucleotides. M21 cytoplasmic extracts (24-h hypoxia) were
used for RNA-UVXL in the absence ( ) or presence of each of the six AS
oligodeoxyribonucleotides (1 µM). RNA-protein complexes
were detected by 8% SDS-PAGE. Background binding in the absence of
extract is indicated as B.
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To further explore the hnRNP L mRNA binding site, we chemically
synthesized the sense oligodeoxyribonucleotide (S1), which was
complementary to AS1 (Fig. 6A)
and attempted to compete for hnRNP L binding to 32P-labeled
HSR. As shown in Fig. 6A, S1 (10 µM)
successfully competed with 32P-labeled HSR for hnRNP L
binding and substantially reduced HSR-hnRNP L complex formation (85%
inhibition of hnRNP L binding). The competition of S1 for hnRNP L
binding to HSR can be seen as low as 0.1 µM AS1 (data not
shown). To define the hnRNP L binding site, eight different
oligodeoxyribonucleotides were synthesized after modification of the
internal base sequences of S1 (Fig. 6). As shown in Fig. 6A,
substitution of base 333 G to A in S1.1 or further substitution of base
335 C to A in S1.2 showed strong competition for hnRNP L binding to the
32P-labeled HSR (S1.1 and S1.2 showed 74 and 82%
inhibition, respectively), thus demonstrating that neither 333 G or 335 C were required for hnRNP L binding. However, substitution of base 337 C to A in S1.3 showed a substantial reduction in the ability to compete
for hnRNP L binding (Fig. 6A, 57% inhibition), and any
modifications after base 337 showed a similar affect (Fig.
6A; S1.4 and S1.5 showed 54 and 50% inhibition,
respectively). These results suggest that bases 332-336 (AGACA) in S1
are not essential for hnRNP L binding and that S1.2 contains the hnRNP
mRNA binding site.

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Fig. 6.
Identification of hnRNP L binding site on
HSR. S1 is a sense-orientated oligodeoxyribonucleotide that is
complementary to AS1 and comprises bases 332-357 of the HSR.
A, competitive RNA-UVXL with sense (S)
oligodeoxyribonucleotides with modifications in the 5'-end. Competitive
RNA-UVXL was performed with 32P-labeled VEGF HSR and M21
cytoplasmic extract (24-h hypoxia) in the absence ( ) or presence of
S1 or its derivatives (10 µM). Band intensities of the
60-kDa HSR-hnRNP L complex were quantified by PhosphorImager analysis.
The percent inhibition was calculated as the percentage decrease in
band intensity compared with control ( ). B, competitive
RNA-UVXL with sense oligodeoxyribonucleotides with 3' modifications.
C, minimal defined 21-base binding site for hnRNP
L-VEGF HSR binding.
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The S1.2 oligodeoxyribonucleotide has three ACAU repeats. To study the
potential importance of the ACAU repeats for hnRNP L binding, three
oligodeoxyribonucleotides (S1.6, S1.7, and S1.8) with 1-3 ACAU repeats
were synthesized (Fig. 6B). As shown in Fig. 6B,
removal of any one of the ACAU repeats from S1.2 substantially reduced
the ability to compete for hnRNP L binding with 32P-labeled
HSR (S1.6, S1.7, and S1.8 showed only 44, 36, and 31% inhibition,
respectively). This result suggests that all three ACAU repeats are
essential for hnRNP L binding. Overall, these results suggest that the
minimal hnRNP L mRNA binding site appears to be
337CACCCACCCACAUACAUACAU357. Using a
GenBankTM Blast search, this 21-base sequence was only
found in human and bovine VEGF 3'-UTRs but not in other VEGF species or
other genes.
The hnRNP L Interacts with VEGF mRNA in Hypoxic Cells in
Vivo--
To determine if hnRNP L interacts with VEGF mRNA
in vivo, we immunoprecipitated hnRNP L with anti-hnRNP L
monoclonal antibody from 24-h hypoxia-cultured M21 cell lysates and
performed RNA extraction, RT-PCR, and Southern blot detection. The VEGF
primers used in RT-PCR contained part of the VEGF coding region and the entire 3'-HSR and should produce a 716-base VEGF165 isoform product. As
shown in Fig. 7A, a unique PCR
product of approximately 700 base pairs in length appeared with hnRNP L
immunoprecipitation, whereas immunoprecipitates for hnRNP C and normal
mouse serum control antibodies did not show any distinct band in the
same region. Southern blot hybridization with VEGF cDNA probe
showed a strong hybridization to the hnRNP L PCR product, thus
confirming the PCR product as VEGF cDNA (Fig. 7B). A
weak interaction of VEGF cDNA probe with the hnRNP C PCR product
was observed, whereas the normal mouse serum control did not have any
detectable signal (Fig. 7B). Studies have shown that hnRNP C
can interact with the poly-U region of mRNAs (30), and thus it is
possible that hnRNP C is associating with many mRNAs including VEGF
in vivo. Taken together, these results verify that hnRNP L
interacts with VEGF mRNA in hypoxic cells in vivo.

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Fig. 7.
Interaction of hnRNP L with VEGF mRNA in
hypoxic cells in vivo. M21 cytoplasmic extracts (24-h
hypoxia) were immunoprecipitated with anti-hnRNP L monoclonal antibody
(L), anti-hnRNP C monoclonal antibody (C), or
normal mouse serum ( ). The co-immunoprecipitated mRNAs were
extracted and used as templates for RT-PCR. The PCR products were
resolved by 1% agarose gel electrophoresis (A) and detected
by Southern blot with a [32P]dCTP-labeled human VEGF
(B).
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Interaction of hnRNP L and VEGF mRNA Is Important for VEGF
mRNA Stability--
To study whether hnRNP L plays an important
role in the regulation of VEGF mRNA expression, M21 cells were
transfected with antisense oligodeoxyribonucleotide AS1 to block
interaction of hnRNP L with VEGF mRNA in hypoxic cells in
vivo. The AS3 oligodeoxyribonucleotide, which did not block the
formation of any HSR-protein complex formation (Fig. 5), was used as
control. As shown in Fig. 8, M21 cells
transfected with AS1 had substantially lower levels of VEGF mRNA
when compared with mock-transfected control cells (54.3% by
PhosphorImager analysis), whereas the GLUT-1 mRNA level was not
affected by the transfection (97.3%), indicating that AS1 is
specifically affecting VEGF mRNA expression. The control
oligodeoxyribonucleotide AS3-transfected cells did not show changes for
either VEGF mRNA (109.5%) or GLUT-1 mRNA (94.5%). These
results suggest that blocking of hnRNP L and VEGF mRNA interaction
in vivo will specifically affect VEGF mRNA accumulation
in hypoxia.

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Fig. 8.
Blocking of hnRNP L and VEGF mRNA
interaction reduces steady state VEGF mRNA level. M21 cells
were transiently transfected in the absence ( ) or presence of 1 µM AS1 or AS3 oligodeoxyribonucleotides, and VEGF,
GLUT-1, and 36B4 mRNA levels were detected by Northern blot
analysis. VEGF and GLUT-1 mRNA signals were normalized with the
ribosome-associated mRNA, 36B4, from each lane and quantified by
PhosphorImager. The mRNA from AS1- and AS3-transfected cells were
calculated as percentage of VEGF or GLUT-1 of mock-transfected mRNA
( ) signals designated as 100%.
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The hnRNPs are a family of abundant nuclear proteins that are involved
in pre-mRNA processing and splicing (22-24). To address whether
the effect of AS1 repression of VEGF mRNA accumulation is through
VEGF mRNA stability, we transfected M21 cells with AS1, AS3, or
mock reagent and examined VEGF mRNA decay under hypoxic conditions.
The rate of decay of the mature VEGF mRNA was determined by
Northern blot hybridization after treatment of cells with the transcriptional inhibitor actinomycin D. As shown in Fig.
9A, M21 cells transfected with
AS1 showed a substantial decrease in VEGF mRNA level after 30 min
of actinomycin D treatment, whereas mock- and AS3-transfected cells
maintained higher VEGF levels for up to 1 h of actinomycin D
treatment. Conversely, GLUT-1 mRNA did not show any changes by AS1
when compared with mock- and AS3-transfected cells (Fig.
9A). When the VEGF mRNA decay curve of a triplicate assay was plotted over a 2-h actinomycin D treatment and VEGF mRNA
half-life (t1/2) was measured, AS1-transfected cells showed a
half-life of 32 ± 5.7 min, whereas mock- and AS3-transfected
cells showed half-lives of 53 ± 4.1 and 57 ± 5.6 min,
respectively (Fig. 9B). AS1-transfected cells showed
statistically significant differences (p
0.0005) in
VEGF mRNA levels when compared with mock-transfected cells at both 30 and 60 min. These results suggest that blocking of the hnRNP L and
VEGF mRNA interaction decreases VEGF mRNA stability under hypoxia. These also suggest that although hnRNP L is important for
mRNA processing, interaction of hnRNP L with VEGF mRNA 3'-UTR plays a critical role in post-transcriptional regulation of VEGF mRNA stability.

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Fig. 9.
Blocking of hnRNP L and VEGF mRNA
interaction by AS1 oligodeoxyribonucleotide decreases VEGF mRNA
stability. M21 cells were transiently transfected in the absence
( ) or presence of 1 µM AS1 or AS3
oligodeoxyribonucleotide, and VEGF mRNA stability was analyzed in
an 8-h hypoxia assay. Actinomycin D (5 µg/ml) was periodically added
beginning at 6 h hypoxia and incubated for 2, 1, or 0.5 h,
respectively, to triplicate plates. Total mRNA was extracted, and
VEGF, GLUT-1, and 36B4 mRNA levels was detected by Northern blot.
A, representative Northern blot analysis of VEGF mRNA
level after actinomycin D treatment. B, VEGF mRNA decay
curves and mRNA half-life (t[itinf,1,2]). VEGF
mRNA signal was normalized with the ribosome-associated mRNA,
36B4, from each lane after quantification by PhosphorImager and
calculated mean ± S.D. from a triplicate experiment. VEGF
mRNA signal without actinomycin D treatment was defined as 100%,
and actinomycin D treated VEGF mRNA levels was calculated as
percentage of decay. The asterisk indicates
p 0.0005 when AS1- and mock ( )-transfected cells
were compared at 30- and 60-min actinomycin D treatment using
Student's t test.
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DISCUSSION |
With a half-life of only 30-45 min under normal growth conditions
(6-8), VEGF mRNA falls within a class of labile mRNAs encoding for many transiently expressed proteins including cytokines,
lymphokines, oncogenes, and transcriptional activators (9). The
mechanisms whereby normally labile mRNAs are stabilized by stimuli
such as hypoxia, growth stimulation, and viral infection are unclear. On the basis of RNA-binding and UV cross-linking, the interaction of
specific cellular proteins with mRNAs have been shown to be altered
in response to stimuli, which correlates with changes in mRNA
stability (9-21). This has led to the hypothesis that mRNA
turnover is mediated principally through mRNA-binding proteins that
specifically recognize AREs and other sequence motifs.
Our previous work demonstrated a 126-base HSR in human VEGF 3'-UTR,
which is responsible for hypoxia-induced VEGF mRNA stability and
forms seven hypoxia-inducible RNA-protein complexes with M21 human
melanoma cell extracts (21). In the present study, we showed that the
protein that forms the 60-kDa RNA-protein complex is present in both
nuclear and cytoplasmic compartments (Fig. 1). Protein sequencing and
immunodepletion studies identified that protein in the 60-kDa
RNA-protein complex as heterogeneous nuclear ribonucleoprotein L (hnRNP
L) (Figs. 3 and 4). The specific mRNA binding site of hnRNP L was
identified as 3'-CACCCACCCACAUACAUACAU-5', a 21-base single-stranded
element, which is unique to human and bovine VEGF 3'-UTR sequences
(Figs. 5 and 6). Immunoprecipitation of hnRNP L followed by RT-PCR
showed that hnRNP L specifically interacted with VEGF mRNA in
hypoxic cells in vivo (Fig. 7). Furthermore, when M21 cells
were transfected with antisense oligodeoxyribonucleotide to the hnRNP L
RNA-binding site, the hypoxia-induced VEGF mRNA half-life decreased
from 53 ± 4.1 min to 32 ± 5.7 min (Fig. 8). This study
identifies for the first time that hnRNP L as a protein present in
human cells that is capable of interacting with VEGF mRNA 3'-UTR
and is functionally involved in the post-transcriptional regulation of
VEGF mRNA under hypoxic conditions.
It is well documented that unstable mRNAs containing AREs generally
consists of either scattered AUUUA pentanucleotides, contiguous AUUUA
repeats, nonamer motif UUAUUUA(U/A)(U/A), or a stretch of AU residues
lacking either motif (10-15). These AREs interact with cytoplasmic
and/or nuclear proteins and usually form ARE-protein complex with
average molecular mass less than 40-kDa (10, 15-20). The VEGF HSR is
also a highly AU-rich element (43% A and 41% U) and contains one
AUUUA pentanucleotide as well as two stretches of AU residues. However,
interaction of the HSR with cellular proteins formed seven RNA-protein
complexes with higher molecular masses (ranging from 40 to 90 kDa). The
HSR also formed same protein complexes with five other cell lines we
tested (data not shown), indicating that those RNA-binding proteins are
common proteins to different cell lines. The 3'-UTRs of two other
hypoxia up-regulated genes, erythropoietin (25) and tyrosine
hydroxylase (26), interacted with mRNA-binding proteins with
apparent molecular masses ranging from 66 to 140 kDa. However, the VEGF
HSR and the 3'-UTRs of both erythropoietin and tyrosine hydroxylase
lack significant homology. Moreover, the interaction site for a 66-kDa
protein to tyrosine hydroxylase 3'-UTR is different from the hnRNP L
mRNA binding site. Thus, VEGF HSR-binding proteins may be distinct from those that recognize other hypoxia-inducible genes.
Studies of rat VEGF mRNA demonstrated a 600-base region covering
nucleotides 1251-1877 in the 3'-UTR required for rat VEGF mRNA
stability (27, 28). However, little homology exists between the human
VEGF HSR sequence and the rat 600-base region. In addition, the protein
complexes observed by EMSA in the rat ARE-binding proteins are 17, 28, and 34 kDa (27, 28), none of which correlate to the hypoxia-induced
complexes described here. Recent studies identified that the 34-kDa rat
VEGF ARE-binding protein as HuR, which interacted with a 45-base
element of rat VEGF 3'-UTR and stabilized the mRNA under hypoxia
(28). This element is also present in the human 3'-UTR covering
nucleotides 1682-1726 3' to the translation stop codon, a region close
to the poly(A) tail. Taken together, these studies suggest that there
are a variety of mRNA-binding proteins present in cells, and the
regulation of mRNA turnover probably depends on the mRNA
sequences, the metabolic and activation state of the cells, and the
regulation of the RNA-binding protein expression and distribution.
The hnRNPs are a family of abundant nuclear proteins that are involved
in pre-mRNA processing and splicing (22-24). The hnRNP L differs
from other ARE-binding hnRNPs in that hnRNP A, hnRNP C, and hnRNP D
interact with the AUUUA motif and may participate in mRNA
destabilization (15, 29-32), whereas hnRNP L interacts with a unique
sequence in the human and bovine VEGF 3'-UTR
(3'-CACCCACCCACAUACAUACAU-5') and probably has a predominant
stabilization effect. In support of this hypothesis, hypoxia
substantially increased hnRNP L in both nucleus and cytoplasmic
compartments, whereas hnRNP C was dramatically decreased to
undetectable levels in the cytoplasm after a 24-h hypoxic treatment.
This differential regulation of hnRNPs under hypoxia, a decrease in the
hnRNP C and an increase in the hnRNP L in cytoplasm, may define a key
balance regulating mRNA metabolism during metabolic stress. The
21-base-long mRNA binding site of hnRNP L is juxtaposed to the only
AUUUA pentanucleotide and a long stretch of AU residues. Therefore, it
is possible that the interaction of hnRNP L may block other proteins
such as hnRNP C from interacting with and destabilizing the mRNA.
Furthermore, under hypoxia we observe increased expression of hnRNP L
and the specific interaction of hnRNP L with VEGF mRNA in
vivo, along with reduced VEGF mRNA stability observed with
transfection of antisense oligodeoxyribonucleotide to the hnRNP L
binding site. Thus, hnRNP L appears to plays a significant role in the
post-transcriptional regulation of VEGF mRNA during hypoxic stress.
Using Western blot analysis, anti-hnRNP L monoclonal antibody
identified three proteins with apparent molecular masses of 66, 60, and
56 kDa, with the cytoplasmic 56-kDa protein being the most strongly
up-regulated by hypoxia. Since there was only one RNA-hnRNP L complex
formed in RNA-UVXL, it is unlikely that all three hnRNP L isoforms
interacted with HSR. Interaction of hnRNP L with the entire 21-base
binding site would account for approximately 6 kDa of the 60-kDa
HSR-hnRNP L complex after UV-cross-linking and RNase treatment. Thus,
it is reasonable to predict that the 56-kDa hnRNP L isoform is the
molecule that is specifically interacting with HSR. Comparing the minor
induction of hnRNP L levels in hypoxia by Western blot to the marked
increase of hnRNP L and HSR interaction in RNA-UVXL assays leads us to
speculate that post-translational modification of hnRNP L isoforms may
promote a higher hnRNP L binding affinity to HSR under hypoxia, an
interesting possibility we are currently investigating.
To effectively define the complex cellular mechanisms that control
mRNA stability such as VEGF, identification of the mRNA-binding proteins is required. Purification and identification of hnRNP L
protein as one of the VEGF mRNA-binding proteins is a significant step in defining the molecular regulation of VEGF mRNA expression under hypoxic conditions. Here we have detailed the characterization of
hnRNP L as one of the hypoxia-inducible RNA-binding proteins that
recognizes a specific region of the human VEGF HSR. The potential signaling mechanisms that regulate hnRNP L isoform expression and
association with VEGF mRNA can now be investigated in detail. Since
a complex of at least seven proteins bind to VEGF HSR, interaction of
hnRNP L with other proteins in the same complex may be required to
protect the VEGF mRNA from nuclease digestion. Interference of
hnRNP L binding to VEGF HSR significantly affected VEGF mRNA expression in hypoxia, indicating that it plays an important and novel
role in VEGF expression under hypoxic stress. Identification of the
other mRNA-binding proteins that recognize VEGF HSR, how they are
regulated, and their potential interaction with hnRNP L will be
necessary to get a complete understanding of the molecular mechanisms
resulting in hypoxia-mediated VEGF mRNA stability.