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INTRODUCTION |
Neuroblastoma (NB)1
accounts for 7-10% of the cancers of childhood with an annual
incidence of ~1/100,000 children under the age of 15 years (1, 2).
Although spontaneous regression frequently occurs in patients with
localized disease (3), the prognosis of tumor stage 3 and 4 (4), which
make up about 50% of the total cases at diagnosis, is generally
unfavorable. NB cells derive from the neural crest and maintain gene
expression profiles and differentiation potentials that are reminiscent
of their embryonic origin (5). A distinctive feature of NB is the
expression of trk receptors on the cell surface that mediate the
differentiation signals sent by neurotrophins (6). Expression of trkA
(7) and trkC (8) is associated with good prognosis, whereas that of
trkB is an indicator of unfavorable outcome (9). As for many other
neoplasms, the molecular mechanisms of NB aggressiveness are not fully
understood. Amplification of the proto-oncogene MYCN and
loss of genetic material at chromosome 1p36 are powerful markers
of poor outcome (10), but the mechanisms through which these
alterations exert their detrimental effects in tumor cells are largely
unknown. The growth of NB cells is dependent on the supply of certain
growth factors that, in the most aggressive forms, are frequently
produced in an autocrine fashion. NB metastases occur in tissues where
a paracrine support of IGF2 is available (11). Non-IGF2-producing
tissues are invaded only by NBs that can autonomously produce IGF2 (12,
13). Thus, the inappropriate expression of some oncogenes can
conceivably lead to the development of growth factor independence via
activation of growth factors or growth factor-regulated pathways.
The biological activity of IGF1 and IGF2 is mediated by a large group
of proteins that constitute the so-called IGF axis composed of
receptors, intracellular substrates (IRS-1 and IRS-2), and transducers
that relay the signal to the nucleus (14). In the extracellular
compartment, another family of proteins, the IGF-binding proteins
(IGFBPs), modulates the action of IGFs. IGFBPs can function as carriers
to transport IGFs in the bloodstream in a ternary complex of 150 kDa
with the acid labile protein (15). In the interstitial space, IGFBP-3
and -5 can bind the extracellular matrix, decrease their affinity for
the IGFs, and exchange them with the IGF1R (16). In other settings,
high concentrations of IGFBPs can inhibit the action of IGFs by
sequestering them from the receptor and cause the onset of apoptosis
(17). The importance of IGFBP-5 in modulating the activity of IGFs is
well recognized in bone and vascular smooth muscle cells (18-20).
IGFBP-5 was also described as an IGF-independent growth factor in bone cells (21), acting through its putative receptor (22). The regulation
of IGFBP-5 expression is finely tuned by a network of stimuli acting
through different transduction pathways (23-26). Noticeably, in smooth
muscle cells, IGF1 is a potent stimulant of IGFBP-5 synthesis through a
transcriptional mechanism involving a signal transduction cascade
dependent on the activation of IGF1R and PI3K/AKT (27, 28). The nuclear
effector(s) and the DNA sequences on the IGFBP-5 promoter
involved in the PI3K/AKT-dependent transactivation of
IGFBP-5 expression are unknown, but it has been hypothesized that an
important role is played by a GC-rich region located between
nucleotides 
147 and 134 from the transcription start (27, 28).
Myb genes (A-myb, B-myb, and c-myb)
are a family of transcription factors whose expression has been
implicated in the control of proliferation and differentiation of
normal and transformed cells (29, 30). For example, c-Myb has been
shown to be essential for the proliferation of normal and leukemic
hematopoietic cells (31) and for the proliferation and survival of
lymphoma cells (32, 33). Although the expression of c-Myb is
predominant in the hematopoietic system, it is also expressed in other
normal tissues (34) and in nonhematopoietic tumors (35, 36).
Interestingly, the expression of IGF1 and IGF1R is stimulated by c-Myb
at the transcription level in fibroblasts (37). The expression of A-Myb and B-Myb occurs in cells of different embryonic origin (38). In
neuroblastoma cells, c-Myb and B-Myb expression is down-regulated during in vitro differentiation (39, 40). Suppression of
c-Myb expression leads to the inhibition of cellular proliferation
(41). Expression of all members of the myb family is
frequent in NB (36), but only that of B-myb appears to be
associated with poor clinical outcome independent of MYCN
amplification and age at diagnosis (36). Together, these findings
suggest that genes of the myb family are important in the
establishment and maintenance of the tumorigenic state of NB cells.
However, information on the cellular targets through which myb genes
exert their function is still scarce. The antiapoptotic ApoJ-1 protein
appears to be a direct transcriptional target of B-Myb in proliferating
neuroblastoma cells (42). Conversely, an atypical role of
c-myb and B-myb in evoking cell death has been
recently described in postmitotic neuronal cells (43). Thus, normal and
transformed neural cells express myb genes that may have
different effects, depending on the physiological or pathological context.
In this study, we investigated whether myb genes regulate
the expression of members of the IGF axis in neuroblastoma cells. Using
a model of c-Myb conditional expression, we found that c-Myb up-regulated the expression of IGF1, IGF1R, and IGFBP-5. c-Myb and
B-Myb transactivated the IGFBP-5 promoter through
binding-dependent and -independent mechanisms. These
findings are discussed in the light of the effects of IGFBP-5 on the
proliferation of NB cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections
Cell lines were cultured in RPMI 1640 medium (LAN-5 cells) (40)
(Euroclone) or in Dulbecco's modified Eagle's medium (N1E-115 cells)
(45) supplemented with 10% fetal calf serum (Hyclone), penicillin and
streptomycin (100 µg/ml each), and 2 mM
L-glutamine at 37 °C, 5% CO2.
For stable transfections, LAN-5 cells were grown at 70% confluence.
Cells were transfected with the tet-responsive vector pBPSTR1 (46)
containing full-length human c-myb cDNA using the calcium phosphate precipitation technique as described previously (45).
Puromycin (Sigma) selection (1 µg/ml) and doxycycline (DOX) (Sigma)
treatment (5 ng/ml) was started 48 h after transfection. Single
clones were expanded after 21 days and tested for inducible c-Myb
expression after removal of DOX.
For transient transfection, murine N1E-115 cells were transfected with
pcDNA3 expression plasmids containing full-length c-myb or B-myb cDNAs or with the empty vector. 48 h
later, cells were harvested, and RNA was extracted for analysis.
Luciferase assays were carried out co-transfecting LAN-5 cells with
reporter vectors pGL3-IGFBP-5-prom wt, M1mut, M2mut,
and M1/M2mut (2 µg) and pcDNA3-c-myb or
-B-myb (1 µg unless otherwise indicated) in the presence
or absence of the mutant K179M AKT plasmid (1 µg) using
the calcium phosphate precipitation technique. The amount of DNA
transfected in each plate was kept constant by adding the appropriate
quantity of empty vector (pcDNA3). 6 h before transfection,
RPMI 1640 medium was replaced with Dulbecco's modified Eagle's
medium. After transfection, fetal calf serum was reduced to 2%, and
luciferase assays were performed 36 h later using a detection kit (Promega).
Plasmids
DBD-c-myb-pcDNA3--
The DNA binding domain of human
c-myb was obtained by PCR using a set of primers containing
the initial ATG (upstream primer) and a stop codon (downstream primer)
after the hemagglutinin tag in-frame with the myb
sequence. The PCR product was then cloned in the EcoRV site
of the pcDNA3 vector.
pGEX-2T-B-myb--
The DNA binding domain of B-myb
was produced in bacteria from vector pGEX-2T-B-myb (42)
using standard techniques.
pBPSTR1-c-myb--
A XhoI/SacII fragment
of 3.4 kb containing the entire coding sequence of human
c-myb was obtained from vector pMbm I/DHFR-c-myb (47) and cloned in the multiple cloning site of the tet-regulated vector pBPSTR1 (46) using standard techniques.
pGL3-IGFBP-5-prom--
The region from nucleotide 459 to +59
from the transcription start was amplified from human genomic DNA by
PCR. The 518-bp product was cloned at the SmaI site of the
pGL3-basic vector (Promega). The sequence of the cloned region was
controlled by dideoxy sequencing.
pGL3-IGFBP-5-prom-
3, -2, and -1--
Deletion mutants
spanning the regions from nucleotides 334 to +59 (3), 209 to +59
(2), and 83 to +59 (1) were generated using the
pGL3-IGFBP-5-prom as template and the following primers: 3 upstream, 5'-GTGTTCACCCTGCTCCGAAGA-3'; 2 upstream,
5'-GAGGGGAGAGGGCGCTGT-3'; and 1 upstream,
5'-GTTGGGAAGCTCAAATTGCAGC-3'.
The common downstream primer for the three deletion mutants was
5'-TTCCAGCGGATAGAATGGCGC-3'.
This primer anneals to nucleotides 121-141 in the pGL3-basic vector.
The PCR-amplified products were digested with HindIII restriction enzyme and cloned in a
HindIII/SmaI-digested pGL3-basic vector using
standard cloning procedures. The sequence of each construct was checked
by dideoxy sequencing.
pGL3-IGFBP-5-prom-M1mut, pGL3-IGFBP-5-prom-M2mut, and
pGL3-IGFBP-5-prom-M1/M2mut--
The three mutants in the M1 and M2
sites were obtained by using the QuikChange site-directed mutagenesis
kit (Stratagene) according to the manufacturer's instructions. The
mutations produced in each site were the same present in the mut M1 and
mut M2 oligonucleotides used in EMSA. The sequence of the mutants was
controlled by dideoxy sequencing.
Dominant Negative K179M AKT and Constitutively Active
Myr-AKT--
The kinase dead dominant negative inhibitor (K179M) of wt
AKT (48) and the constitutively active Myr-AKT (49) were
kindly obtained by Dr. Tsichlis (Thomas Jefferson University,
Philadelphia, PA).
RNA Analysis
Total RNA was prepared by Trizol extraction (Invitrogen).
Carryover DNA contamination was eliminated by treatment of the total RNA with the DNA-free kit (Ambion) according to the manufacturer's instructions. RNA was reverse-transcribed with the first-strand cDNA synthesis kit for reverse transcription-PCR (Roche Molecular Biochemicals) using an input of 500 ng for each reaction. Subsequent PCRs were carried out for the indicated number of cycles at the appropriate annealing temperature for each pair of primers. Northern blot analysis was carried out as described previously (50) using standard techniques.
Plasmids Containing the Sequences Used as Probes for Northern
Blot Analysis
Probes for IGFBP-5 and IGF1R were obtained
by PCR amplification of human genomic DNA and cloning the amplified
products in the pGEM-T vector (Promega). The primers used to amplify
IGFBP-5 were as follows: upstream primer,
5'-TAGTGCCCTCAACTCTCTGG-3'; and downstream primer,
5'-GGGACGCATCACTCAACGTT-3'. The primers used to amplify
IGF1R were as follows: upstream primer,
5'-ACAACTACGCCCTGGTCATC-3'; and downstream primer,
5'-TGGCAGCACTCATTGTTCTC-3'. The probe for c-myb was obtained
from vector Bluescript-c-myb, in which the first 1200 nucleotides from the 5'-end of human c-myb cDNA had been
cloned previously in our laboratory. The IGF2 probe was
obtained from vector pB4 (51). The IGF1 probe was obtained
from vector pPI-IGF1 (a gift of Dr. Maria Giulia Rizzo,
Regina Elena Institute, Rome, Italy) containing the coding
region of the gene. The 
-actin probe was obtained from
vector HfA-1 (52).
Protein Analysis
Cellular proteins were extracted as described previously (45).
Briefly, cells were lysed on ice in 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 250 mM NaCl, 50 mM NaF,
0.1% Triton X-100, 0.1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml
leupeptin. Lysate was then centrifuged at 14,000 × g
for 10 min at 4 °C, the supernatant was collected, and protein
concentration was determined using a colorimetric assay (BioRad).
Protein extraction from the culture supernatants was carried out as
follows: cells were plated in normal medium, and 24 h later, the
medium was replaced with 2.5 ml of Dulbecco's modified Eagle's medium
without fetal calf serum. After keeping the cells in culture for
40 h, the supernatant was collected, supplemented with 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, and
10 mg/ml aprotinin, and centrifuged for 10 min at 1500 rpm at 4 °C.
The supernatant was transferred to a Centricon 30 (Amicon) and
centrifuged for 1 h and 15 min at 5000 rpm at 4 °C. The
concentrated sample was collected by inverting the Centricon 30 device
and centrifuging for 2 min at 3500 rpm at 4 °C. Protein separation
on SDS-polyacrylamide gels and Western blot analysis were carried out
as described previously (45). Detection was performed by using the ECL
Plus kit (Amersham Biosciences) following the manufacturer's instructions.
Antibodies
Polyclonal anti-B-Myb (c-20) and anti-IGFBP-5 (c-18) antibodies
were purchased from Santa Cruz Biotechnology. Monoclonal anti-c-Myb antibody (clone 1-1) was purchased from Upstate Biotechnology; monoclonal anti-hemagglutinin antibody (clone 12CA5) was purchased from
Roche Molecular Biochemicals; monoclonal anti--actin (clone AC-15)
was purchased from Sigma.
EMSA and Chromatin Cross-linking Immunoprecipitation
Murine neuroblastoma N1E-115 cells transfected with the
expression vector containing the DNA binding domain of human
c-myb or the empty vector were collected and lysed in 20 mM Hepes, pH 7.9, 0.4 M NaCl, 25% glycerol, 1 mM EDTA, 2.5 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride on ice for 20 min, frozen, thawed, and centrifuged for 10 min at 13,000 × g at
4 °C. Supernatants were collected and stored at 80 °C until
use. EMSAs were carried out as described previously (40). Briefly,
binding reactions were done in 10 µl of 20 mM Tris-HCl,
pH 7.5, 3 mM MgCl2, 50 mM NaCl,
15% glycerol, 0.2 µg/µl poly(dI-dC), 15 µg of cellular extract, and 1 × 105 cpm of double-stranded oligonucleotide
(see below). In competition experiments, 5 pmol of the double-stranded
competitor oligonucleotides were added to the mix. The reactions were
incubated for 10 min on ice and loaded on a 6% polyacrylamide gel in
0.25× Tris-borate EDTA at 140 V.
EMSAs to detect B-Myb binding were carried out as described above using
300 ng of the recombinant glutathione S-transferase-B-Myb protein.
The MBS-1 oligonucleotide containing the consensus Myb recognition
element was 5'-CTCTACACCCTAACTGACACATTCT-3'. The M1 oligonucleotide was
5'-AATTGCAGCTACAAACTGGCTGGCAGCCAG-3'. The mut M1 in which the M1
binding site was mutated (underlined) was
5'-AATTGCAGCTACGCCTTGGCTGGCAGCCAG-3'. The M2
oligonucleotide was 5'-GCACGGTATATCCAGTTGGCTAATAAGAAA-3'. The mut M2 in
which the M2 binding site was mutated (underlined) was
5'-GCACGGTATATCCAAGGCGCTAATAAGAAA-3'.
Chromatin cross-linking immunoprecipitation was carried out essentially
as described previously (53), with few modifications. We started with
20 × 106 cells for each experimental point.
Sonication was carried out at 4 °C with 12 pulses of 2 s each
(35% amplitude) using a Sonics Vibracell 500 W equipped with a
microtip. DNA size in the range from 2000 to 600 bp was monitored on an
agarose gel. Immunoprecipitations using protein A-agarose (Roche
Molecular Biochemicals) were carried out using 4 µg of each antibody
for each experimental point. At the end of the procedure, DNA from each
point was amplified by PCR using the following primers: upstream M1
primer, 5'-CCCGTGTGAGTTTGTACTGC-3'; and downstream M1,
5'-CACACTGCTTTGCAGCTCTTT-3'. The PCR annealing temperature was
53 °C. Reactions were carried out in the presence of 5%
Me2SO in PCR mix. This set of primers amplifies the
region from nucleotide 135 to +59. Upstream M2 primer was
5'-GCTTAGGAAGATTTCTTGGGC-3'. Downstream M2 primer was
5'-GCTACCGAGAATGGGGAGG-3'. This set of primers amplifies the region
from nucleotide 459 to 266. Amplification products were run on an
agarose gel and transferred to nylon membrane (Hybond N+; Amersham
Biosciences). Membranes were hybridized as described previously (41)
using the following 32P-labeled probes: M1 probe,
5'-GGGAAGCTCAAATTGCAGCT-3', spanning the region between nucleotides
81 and -61; and M2 probe, 5'-CCTCATTGTGTTCACCCTGC-3', spanning the
region between nucleotides 341 and -321.
Proliferation Assays
Cell proliferation was measured using the colorimetric cell
proliferation kit II (WST-1; Roche Molecular Biochemicals) based on the
colorimetric detection of a formazan salt. In each well, 4 × 104 cells were seeded in RPMI 1640 medium supplemented with
2% heat-inactivated fetal calf serum. Recombinant IGFBP-5 (Upstate
Biotechnology, Inc.) and IGF2 (Sigma) were added at the
indicated concentration, and the colorimetric reading at 450 nm was
carried out after 40 h according to the manufacturer's
instructions. Background absorbance of each sample at 630 nm was
subtracted from the readings at 450 nm.
| |
RESULTS |
IGFBP-5 Expression Increases in Neuroblastoma Cells Expressing
c-myb and B-myb--
The human neuroblastoma cell line LAN-5 has been
studied extensively for its ability to differentiate along a neural
pathway upon exposure to all-trans-retinoic acid
(40). These cells produce low but detectable amounts of both
c-Myb and B-Myb, whose expression is down-regulated during
all-trans-retinoic acid-induced differentiation (39, 40).
LAN-5 cells were used to generate stable transfectants conditionally
expressing c-Myb. c-myb full-length cDNA was
cloned into the tet-regulated plasmid pBPSTR1 (46), and the
resulting construct was used to transfect LAN-5 cells. After puromycin
selection, resistant clones were selected for their ability to express
increased amounts of c-Myb after withdrawal of the tetracycline
homologue DOX from the media. One clone, referred to hereafter as
LAN-5-c-myb, expressed high levels of c-Myb in the absence of DOX.
Expression of some members of the IGF axis, whose activation is highly
relevant for survival of neuroblastoma (11, 54), was tested in the presence or absence of DOX. Upon c-Myb expression, there was an increase in the mRNA levels of IGF1, IGF1R,
and IGFBP-5 (Fig. 1A). Up-regulation of
IGF1 and IGF1R was in part expected because it
has been demonstrated previously that c-Myb induces an increase in the
expression of these genes in fibroblasts (37). On the contrary, the
strikingly elevated expression of IGFBP-5 represented a
novel and unexpected finding. Of interest, the electrophoretic pattern
of IGF2 RNA differed in the presence and absence of
DOX (Fig. 1A). IGF2 is transcribed from distinct
promoters giving rise to transcripts of different lengths ranging from
2.2 to 6.0 kb (55). In the absence of c-Myb induction
(+DOX), there is expression of the 6.0- and 4.8-kb mRNA
forms that arise from use of the P2 and P3 promoters, respectively
(Fig. 1A). Upon c-Myb induction (DOX), it
appears that there is a preferential use of the P3 promoter from which
the 4.8-kb RNA arises (Fig. 1A). Thus, IGF2,
compared with IGFBP-5, IGF1, and
IGF1R, is not up-regulated by c-Myb expression in
neuroblastoma cells.
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Fig. 1.
IGFBP-5, IGF1R,
and IGF1 are up-regulated after c-Myb induction
in human neuroblastoma cells. A, Northern blot
analysis of LAN-5-c-myb cells before and after DOX withdrawal. The blot
was hybridized with probes specific for the genes indicated on the
left. The size of the predominant mRNA species is
indicated on the right. B, Western blot
analysis of LAN-5-c-myb cells before and after DOX withdrawal. The
top blot represents an equal amount of culture supernatant
after protein concentration probed with a specific anti-IGFBP-5
antibody. Bottom blots were carried out on cellular extracts
using specific anti-c-Myb and anti--actin antibodies.
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To demonstrate that the c-Myb-induced increase of IGFBP-5
mRNA levels correlated with enhanced protein expression, culture supernatants were prepared from LAN-5-c-myb cells in the presence or
absence of DOX and tested by Western blot analysis for IGFBP-5 expression. As shown in Fig. 1B, an increased amount of
IGFBP-5 protein was detected in the supernatant of LAN-5-c-myb cells
after c-Myb induction (Fig. 1B), in good correlation with
the increased expression of c-Myb in the corresponding cellular
extracts (Fig. 1B).
One possible problem with these results is that they could reflect the
clonal nature of the LAN-5-c-myb cell line. In other words, the
observed differences could be intrinsic to this particular cell clone.
Thus, we transiently transfected the murine neuroblastoma cell line
N1E-115 with expression vectors for c-myb or
B-myb. 40 h after transfection, cells were collected,
and total RNA was prepared. Identical amounts of RNA from each
experimental point were reverse-transcribed, and cDNAs were
PCR-amplified for 25, 30, and 35 cycles using specific primers to
detect c-myb, B-myb, and IGFBP-5 (Fig.
2A). After normalization for
-actin levels, the intensity of each band was analyzed
and expressed in densitometric units, taking the intensity of the
control as 1 (Fig. 2B). Cells transfected with
c-myb and B-myb expressed approximately double the amount of IGFBP-5 expressed by control cells transfected
with the pcDNA3 vector alone. It should be noticed that the
increase in IGFBP-5 expression is probably underestimated
because the transfection efficiency of N1E-115 cells was ~30% (data
not shown). Thus, the increase of IGFBP-5 expression in
neuroblastoma cells expressing c-Myb and B-Myb is not restricted to a
particular cell clone.
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Fig. 2.
IGFBP-5 induction by c-Myb and
B-Myb in mouse neuroblastoma cells. A,
semiquantitative reverse transcription-PCR on RNA from N1E-115 cells
transfected with empty vector or c-myb and B-myb
expression vectors. Reverse transcription-PCRs were carried out 48 h after transfection using a pair of primers specific for each of the
genes indicated at the below the panel for the indicated
numbers of PCR cycles. B, quantitative densitometric
measurements of IGFBP-5 mRNA expression in empty
vector-, c-myb-, and B-myb-transfected N1E-115
cells after 30 cycles of PCR. Expression of IGFBP-5 in empty
vector-transfected cells was arbitrarily set to 1.
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c-Myb and B-Myb Transactivate the Proximal Region of the Human
IGFBP-5 Promoter--
Analysis of the proximal region of the human
IGFBP-5 promoter revealed two bona fide Myb
binding sites at positions 59 to 54 (hereafter termed M1) and 429
to 424 (M2). It is noteworthy that a potential Myb/E-box site,
involved in transcriptional inhibition by cortisol in bone cells, was
mapped in the mouse promoter at a position corresponding to M1 (23). A
similar sequence was also found in the rat IGFBP-5 promoter,
where it mediated responsiveness to osteogenic protein-1 (57). In the
human promoter, the first base at the 5'-end of the core M1
sequence is a purine (A) instead of a pyrimidine as in the canonical
Myb recognition element. The M2 site contains a perfect core consensus
element in opposite orientation with respect to the direction of the
transcription. The region spanning nucleotides 459 to +59 of the
human IGFBP-5 promoter was cloned in the reporter vector
pGL3-basic upstream of the luciferase gene (pGL3-IGFBP-5-prom), and
functional assays were carried out in LAN-5 cells transfected with a
fixed amount (2 µg) of the reporter vector (pGL3-IGFBP-5-prom) and
increasing amounts (from 0.5 to 2 µg) of expression vectors coding
for human c-myb or B-myb. Both genes
transactivated the IGFBP-5 promoter, albeit with different
efficiency (Fig. 3, A and
B). B-Myb was more effective than c-Myb when transfected in
amounts up to 1 µg. Thus, the human IGFBP-5 promoter is
transactivated by c-Myb and B-Myb.
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Fig. 3.
c-Myb and B-Myb transactivate the human
IGFBP-5 promoter. Human neuroblastoma LAN-5 cells
were transfected with a fixed amount of the reporter vector
pGL3-IGFBP-5-prom (2 µg) and increasing amounts (0.5, 1, and 2 µg)
of c-myb (A) or B-myb (B)
expression vectors. Each experiment was carried out in triplicate.
Bars, ±S.D.
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c-Myb and B-Myb Bind the M1 and M2 Sites in Vitro and in Vivo in
the Human IGFBP-5 Promoter--
To determine whether the M1 and M2
sites could interact with Myb proteins, we carried out EMSAs using
cellular extracts from N1E-115 neuroblastoma cells transfected with an
expression vector carrying the DNA binding domain of human c-Myb and
oligodeoxynucleotides including the potential Myb binding sites M1 and
M2. Myb proteins bound to the M1 and to M2 sites (Fig.
4), and the interaction was efficiently
competed by an excess of wt M1 or M2 or by the control MBS-1 containing
a canonical Myb recognition element (40), but not by an excess of mut
M1 or mut M2 oligodeoxynucleotides in which the Myb binding site was
mutated. Although the amount and the specific activity of the labeled
M1 and M2 oligodeoxynucleotides were equivalent, the retarded complex
formed with radiolabeled M2 was more abundant than that detected with
M1. This suggests that c-Myb has a higher affinity for M2 than for M1,
at least in vitro. In experiments carried out using a
glutathione S-transferase-B-Myb fusion protein containing
the DNA binding domain of B-Myb, we observed similar results
demonstrating the ability of B-Myb to bind M1 and M2 in
vitro (data not shown).
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Fig. 4.
c-Myb and B-myb bind in vitro
to M1 and M2 sites in human IGFBP-5
promoter. Band-shift experiments were carried on N1E-115
cellular extracts transfected with c-myb expression vector in the
presence of 32P-labeled wt M1 and M2 double-stranded
oligonucleotides. Competitions were performed using a 300-fold excess
of cold wt and mutant double-stranded oligonucleotides. An
oligonucleotide containing a canonical Myb binding site
(MBS-1) was used as a positive control. Specific retarded
bands (ac) are indicated to the left of each
blot.
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A limitation of the EMSA technique resides in the marked difference
between the binding of a transcription factor to an
oligodeoxynucleotide in an acellular context and the binding of the
same factor in the complexity of the cellular microenvironment.
Recently, these problems have been overcome by developing the chromatin
cross-linking immunoprecipitation technique (53), which detects the
binding of a specific protein to the DNA in living cells. We used the LAN-5 neuroblastoma cell line to test the binding of c-Myb to M1 and M2
sites in the promoter of the IGFBP-5 gene in
vivo; these cells express moderate amounts of c-Myb and B-Myb
(36, 45), possibly representing the levels found in most
neuroblastomas. A c-Myb-specific antibody was used to immunoprecipitate
the cross-linked chromatin from cycling LAN-5 cells in normal culture
conditions. After amplification of two regions containing the M1 and M2
sites, electrophoretic separation, blotting, and hybridization to
specific probes detected the binding of c-Myb to both sites (Fig.
5, left blots). On the
contrary, no bands were visible in amplifications carried out with
chromatin immunoprecipitated with an unrelated antibody and in the
control with no antibody. A similar chromatin cross-linking
immunoprecipitation experiment was carried out using a B-Myb-specific
antibody. Also in this case we detected the binding of B-Myb to M1 and
M2 sites (Fig. 5, right blots).
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Fig. 5.
c-Myb and B-Myb bind in vivo
to M1 and M2 sites in human IGFBP-5
promoter. Chromatin cross-linking immunoprecipitation was
carried out on proliferating LAN-5 cells using antibodies specific to
c-Myb (left panels) or B-Myb (right panels).
No Ab, immunoprecipitations in the absence of
antibodies; Mock, reactions in the absence of chromatin
and antibodies. PCRs on the immunoprecipitated chromatin were carried
out for 25 cycles, and amplification products were blotted and
hybridized with specific probes for the amplified regions.
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Transactivation of IGFBP-5 by Myb Occurs through Two Distinct
Mechanisms--
To assess the function of M1 and M2 sites in
IGFBP-5 transcriptional control, we generated by
site-directed mutagenesis three mutant forms of the pGL3-IGFBP-5-prom:
mut M1 (in which the M1 site was mutated), mut M2 (in which the M2 site
was mutated), and mut M1/M2 (in which both M1 and M2 were mutated). In
addition, we also generated three deletion mutants of the
IGFBP-5 promoter. A schematic representation of the
IGFBP-5 promoter with the most relevant sites, the mut M1,
mut M2, and mut M1/M2 and the deletion mutants 1, 2, and 3 are
shown in Fig. 6A. These
constructs were used in functional assays carried out co-transfecting
the LAN-5 cell line with fixed amounts of each reporter (2 µg) and the expression vector (1 µg) coding for c-Myb or B-Myb. The
transactivation of the wt and mutant IGFBP-5 promoters by
c-Myb or B-Myb was normalized with respect to their controls
(i.e. co-transfections of the reporter and the empty
vector). Compared with the effect of c-Myb and B-Myb on the wt
IGFBP-5 promoter, the M2 mutation caused a decrease in
reporter gene transactivation, whereas the M1 mutant was transactivated more efficiently (Fig. 6B). This result suggests that the
binding of Myb to the M2 site has a stimulatory effect on
IGFBP-5 transcription. In contrast, Myb binding to M1 seems
to cause transcriptional repression. The mut M1/M2 promoter was
transactivated as effectively as the wt promoter (Fig. 6B).
Because we found an increase in the expression of both IGF1
and IGF1R after c-Myb induction (Fig. 1A), the
up-regulation of IGFBP-5 in neuroblastoma cells could occur at least in
part through an indirect mechanism involving activation of IGF1R and
its downstream effectors PI3K/AKT (28). To test this hypothesis, we
transfected LAN-5 cells with the wt IGFBP-5 promoter and a
constitutively active form of AKT (myr-AKT) (49). Transcription from
the IGFBP-5 promoter was increased ~2-fold by myr-AKT
(Fig. 7A). On the contrary,
the specific PI3K/AKT pathway inhibitor LY294002 was able to inhibit
transcription below the basal level (Fig. 7A).
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Fig. 6.
Effects of Myb in the transactivation of the
IGFBP-5 promoter. A, schematic
representation of the proximal region of the human IGFBP-5
promoter and of the mutant plasmids used in functional assays of
promoter activity. B, luciferase assays were carried
out on LAN-5 cells transfected with c-myb (left
panel) or B-myb (right panel) expression
vectors and wt or mutant IGFBP-5 promoter reporter vectors
as indicated. Fold activation was calculated, taking the level of
luciferase activity of each reporter vector co-transfected with the
empty vector as 1 ( ). Experiments in B were carried out
in triplicate. Bars, ±S.D.
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Fig. 7.
AKT-mediated transactivation of the
IGFBP-5 promoter. A, LAN-5 cells
were co-transfected with wt pGL3-IGFBP-5-prom (wt) and
pcDNA3 empty vector or with a constitutively active AKT
expression vector (myr-AKT) or treated with LY294002 at the indicated
concentration for 3 h before the assay. B,
luciferase assays were carried out on LAN-5 cells constitutively
expressing c-Myb with wt or mutant IGFBP-5 promoter reporter
vectors as indicated plus or minus AKT K179M. The luciferase activity
of each reporter in the absence of AKT K179M was set to 100, and the
percentage of luciferase activity in the presence of AKT K179M was
calculated accordingly. C, LAN-5 cells were
co-transfected with the wt pGL3-IGFBP-5-prom or the deletion mutant
1, 2, or 3 and expression vectors for myr-AKT or
c-myb. Fold activation was calculated taking the promoter
activity of the wt pGL3-IGFBP-5-prom co-transfected with the empty
vector pcDNA3 as 1. Experiments in AC were carried out
in triplicate. Bars, ±S.D.
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Thus, we tried to uncouple direct and indirect effects of Myb on
transactivation of the IGFBP-5 promoter using a kinase dead dominant negative inhibitor (K179M) of wt AKT (48). In this case,
functional assays were carried out in a neuroblastoma cell line
constitutively overexpressing c-Myb. Cells were transfected with a
fixed amount of the reporter vector (1 µg) with or without the K179M
AKT plasmid (3.5 µg). When necessary, the amount of transfected DNA
was kept constant by adding an appropriate amount of the pcDNA3
empty vector. The transactivation activity of each reporter construct
was set to 100 (Fig. 7B, 
). The relative residual activity of each reporter construct in presence of the dominant negative K179M AKT plasmid was calculated accordingly (Fig.
7B, ). Interestingly, the reporter activity driven
by the mut M2 (with the intact M1 site) or the double mutant mut M1/M2
promoter was markedly reduced upon expression of the K179M AKT plasmid. On the contrary, the effect of the K179M AKT plasmid on the wt or on
the mut M1 promoter (with the intact M2 site) was less dramatic. Together, these data indicated that M2 is the only site required for
Myb-dependent transactivation of the IGFBP-5
promoter. Moreover, the drastic decrease in transactivation in the
presence of the K179M AKT plasmid using the mut M1/M2 reporter strongly
suggests that Myb transactivation of the IGFBP-5 promoter is
partly dependent on an indirect mechanism mediated by AKT.
To determine which regions of the IGFBP-5 promoter are
directly involved in the AKT-dependent transactivation,
three deletion mutants spanning nucleotides 334 to +59 (3), 209
to +59 (2), and 83 to +59 (1) were generated in pGL3-basic
reporter vector. A graphic representation of these constructs is shown
in Fig. 6A. LAN-5 cells were transfected with these mutants
and c-myb expression vector. Transactivation by c-Myb is
attenuated in luciferase assays with the 3 promoter, which lacks the
functional M2 site (Fig. 7C). In addition, c-Myb is no
longer able to transactivate 2 and 1 truncated promoters (Fig.
7C). In parallel experiments, we studied the effect of
constitutively active AKT (myr-AKT) on the same mutants (Fig.
7C). The 3 promoter was transactivated by AKT as
effectively as the wt promoter. Of interest, the 3 mutant was
similarly transactivated by AKT and by c-Myb, suggesting that in the
absence of the M2 site, transactivation by c-Myb depends largely on AKT
stimulation. A significant loss in AKT responsiveness was observed in
luciferase assays carried out with the 2 mutant, whereas the 1
mutant was no longer responsive to AKT. Thus, the AKT-responsive
element(s) is located in the region spanning the 3 and 1 IGFBP-5 promoter.
IGFBP-5 Protein Affects Proliferation of Neuroblastoma
Cells--
In bone and vascular smooth muscle cells (18-20), IGFBP-5
modulates cellular proliferation. To assess the relevance of IGFBP-5 expression in neuroblastoma cells, experiments based on a colorimetric assay were carried out to measure proliferation in the presence of
increasing amounts of recombinant IGFBP-5 protein in LAN-5 cells that
express low amounts of endogenous IGFBP-5. Cells were cultured in 2%
heat-inactivated fetal calf serum plus 0.1, 1, 100, and 200 ng/ml human
recombinant IGFBP-5 for 48 h. Under these conditions, an increase
(6.3 ± 3.8%) in proliferation was detected (Table
I) at a 1 ng/ml concentration of
IGFBP-5, followed by a decrease (10.7 ± 3.4%) at 200 ng/ml;
both variations were statistically significant. We also cultured the
cells in the presence of a fixed quantity of recombinant IGF2 (100 ng/ml), the most relevant IGF in neuroblastoma (11), together with an
increasing concentration of IGFBP-5 (0.1-100 ng/ml). A dramatic
increase in proliferation (209.7 ± 44.9%) was observed at 1 ng/ml IGFBP-5 (Table II). The proliferation returned at a level similar to that of control cells at a
concentration of 100 ng/ml IGFBP-5. Thus, IGFBP-5 is effective in
modulating proliferation of neuroblastoma cells.
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Table I
Effect of IGFBP-5 on proliferation in LAN-5 human neuroblastoma
cells
The experiment was carried out in triplicate cultures (40,000 cells/well). Values are means ± S.D. represents the
percentage difference in proliferation with respect to that calculated
in the absence of recombinant IGFBP-5. Statistical significance (p)
with respect to the value in the absence of recombinant IGFBP-5 was
calculated by the two-tailed Student's t test.
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Table II
Effect of IGFBP-5 in presence of ectopically added IGF2 in LAN-5 cells
The experiment was carried out in sextuplicate cultures (40,000 cells/well). Values are means ± S.D. represents the percentage of
difference in proliferation with respect to that calculated in the
absence of recombinant IGFBP-5. Statistical significance (p) with
respect to the value in the absence of recombinant IGFBP-5 was
calculated by the two-tailed Student's t test.
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DISCUSSION |
Previous studies have implicated IGFs in the growth and survival
of NB cells (11, 54). The role of myb genes in controlling the expression of IGF1 and IGF1R has been
demonstrated in fibroblasts (37). Using a NB cell line conditionally
expressing c-Myb, we show here that the
c-Myb-dependent up-regulation of IGF1 and
IGF1R is not limited to fibroblasts. More importantly, we
have identified a new Myb target gene, IGFBP-5, which belongs to the
group of proteins that bind IGF with high affinity (60, 61). Myb
proteins appear to control IGFBP-5 expression at the transcriptional
level. This conclusion is corroborated by several observations. The
analysis of the proximal region of the human IGFBP-5
promoter revealed two potential Myb binding sites at positions 59 to
54 (M1) and 429 to 424 (M2) from the transcription start site.
The most proximal site, M1, is located in close proximity to a
TATA-like sequence in a region with a high degree of conservation among different species. In humans, M1 is mutated in the first base of the
core sequence, which is a purine instead of a pyrimidine as in the
canonical Myb binding site. M2 is located in a less conserved region,
although a similar site in the same orientation was found at
nucleotides 351 to 346 of the mouse gene. c-Myb and B-Myb were able
to bind to both M1 and M2 sites in vitro and in
vivo, as revealed by EMSA and by chromatin cross-linking
immunoprecipitation. In vitro, Myb binding to M2 was
stronger than Myb binding to M1, suggesting a partial impairment in
binding due to the above-mentioned mutation in the first base of the
core sequence of M1. In vivo, we were unable to detect a
similar difference in binding to M1 and M2. This discrepancy could be
due to insufficient discrimination of high and low affinity binding
sites of our chromatin cross-linking immunoprecipitation procedure or
to different binding affinities of Myb proteins in vitro and
in vivo. In fact, it has been reported that transcription
factors that interact weakly with binding sites in vitro can
bind with higher affinity in vivo (53, 62).
In functional assays, c-Myb and B-Myb were able to transactivate the
IGFBP-5 promoter in NB cells. B-Myb was a better
transactivator than c-Myb, possibly because of its ability to act
through a binding-independent mechanism in cooperation with the Sp1
factor (63). In agreement with the latter hypothesis, potential
Sp1 binding sites were described in the proximal region of the
IGFBP-5 promoter (27, 28). Mutations of the M1 and M2 sites
and use of deletion mutants revealed a high complexity in the
Myb-dependent regulation of the IGFBP-5 promoter. In this regard, mutation in M2 brought about a decrease in
transactivation by Myb proteins, and the deletion mutant 3, which
lacks the region containing M2, was less responsive to c-Myb transactivation. In contrast, mutation of the M1 site revealed an
increased transactivation by c-Myb or B-Myb. Moreover, c-Myb transactivation was also abrogated using the deletion mutants 2 and
1, in which only the M1 site was still present. These results
suggest functionally different roles for the M1 and M2 sites. M2 acts
as as a positive cis-regulatory element. In contrast, M1,
located in close proximity to a TATA-like sequence (34 to 25 from
the transcription start) in the IGFBP-5 promoter, does not
act as an activator site upon Myb binding. Of interest, a Myb binding
site overlapping the TATA box in the c-erb-2 promoter was
shown to cause transcriptional repression upon c-Myb or B-Myb binding
(65). The ability of c-Myb to transactivate the mut M2 and the mut
M1/M2 promoters suggested the existence of Myb binding-independent
mechanisms of IGFBP-5 promoter regulation. Because Myb
expression stimulated IGF1 and IGF1R in NB, and
the PI3K/AKT pathway appears to play a role in IGFBP-5
transactivation, part of the effect induced by c-Myb on the
IGFBP-5 promoter could be through the IGFR1 via the PI3K/AKT
pathway (28). In agreement with this hypothesis, the IGFBP-5
promoter was transactivated by a constitutively active AKT (49),
whereas expression of a dominant negative AKT mutant (48, 66) markedly
suppressed Myb-dependent transactivation of the mut M2 and
mut M1/M2 promoters. Of interest, dominant negative AKT was markedly
less effective on the wt and mut M1 promoters that retained a
functional M2 site activated by c-Myb via a direct
binding-dependent mechanism. The wt and the 3 deletion
promoter (which lacks the M2 site) were comparably activated by AKT,
further suggesting that the effect of AKT is independent of the
functional Myb binding site. The responsiveness to AKT was
significantly decreased in the 2 promoter and was completely lost in
1. Taken together, these results suggest that the AKT-responsive
region is located between nucleotides 334 and 83 in the human
IGFBP-5 promoter. Thus, transcription of IGFBP-5
appears to be subjected to direct regulation via Myb binding and to
Myb-dependent indirect activation through the AKT pathway.
Besides IGF1/IGF1R (28), interleukin 6 with its soluble receptor (67),
prostaglandin E2 (26), and retinoic acid (24) stimulate the
expression of IGFBP-5 messenger RNA. Conversely, osteogenic
protein-1 down-regulates the transcription of IGFBP-5 in
primary culture of fetal rat calvaria cells through a 21-bp control
element that includes the rat homologue of the M1 site (57). A similar
inhibitory effect is brought about by basic fibroblast growth factor,
cortisol (23), dexamethasone (68), platelet-derived growth factor (64),
and transforming growth factor-, which acts through a c-Jun
N-terminal kinase-dependent pathway (59). Such an intricate
mechanism of control is not surprising in light of the delicate
biological function of IGFBP-5. In fact, depending on the amount of the
protein, the microenvironment, and the cell type, IGFBP-5 can act as
positive or negative regulator of cellular proliferation and
apoptosis in association with or independently of IGFs (17, 22). It
should be stressed that our findings on Myb-dependent
transactivation of IGFBP-5 promoter apply, at present, only
to NB cells, given the well known tissue-specific regulation of IGFBPs
(56, 58).
NB preferentially metastasizes in cellular districts where a paracrine
supply of IGF2 is available (11). Aggressive NBs frequently acquire an
autocrine loop of IGF2 production that sets the tumor cells free from
external sources (12). These findings underscore the importance of IGFs
and, in general, of the activation of the IGF axis in NB. In this
study, we demonstrated the effect of IGFBP-5 expression on the
proliferation of neuroblastoma cells. The proliferation activity
induced by ectopically added IGF2 can be strongly enhanced by IGFBP-5.
Notably, it has been demonstrated that IGFBP-5 binds preferentially
IGF2 (44). In light of the IGF2 dependence of NB, the IGFBP-5 role in
enhancing IGF2 activity can be regarded as a further advantage acquired
by tumor cells. On the other hand, the effects of IGFBP-5 were
dose-dependent, because when it was added in high
concentrations, the recombinant IGFBP-5 protein caused a decrease in
proliferation. Thus, a cell can be pushed toward proliferation or
doomed to apoptosis, depending on the prevalence of one or another
member of the IGF axis. In this scenario, myb genes seem to
play a central role in the regulation of IGF signaling.
In summary, our data add a piece of information to the complex
mechanism of IGFBP-5 transcriptional regulation that
involves Myb-dependent direct (via binding sites) and
indirect (via an AKT-responsive promoter region) mechanisms (Fig.
8). Furthermore, the notion that
IGFBP-5 and IGF1 and IGF1R are targets
of Myb in NB cells provides intriguing hints on the cellular targets activated by these transcription factors to promote the process of
neoplastic transformation.
§,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.:
39-063048-3172; Fax: 39-063048-6559; E-mail:
raschella@casaccia.enea.it.
