From the Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires, CNRS UPRES-A 7053, Université Paris XII, Avenue du Général de Gaulle, 94010 Créteil Cedex, France
Received for publication, December 4, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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Heparin affin regulatory peptide (HARP) is
a 18-kDa heparin-binding polypeptide that is highly expressed in
developing tissues and in several primary human tumors. It seems to
play a key role in cellular growth and differentiation. In
vitro, HARP displays mitogenic, angiogenic, and neurite outgrowth
activities. It is a secreted protein that is organized in two Heparin affin regulatory peptide
(HARP)1 (1) is a secreted
protein that belongs to the superfamily of heparin-binding growth factor. Also referred to as pleiotrophin (2), heparin-binding growth-associated molecule (3), and osteoblast specific factor-1 (4),
HARP shares 50% of homology with midkine (5, 6) and both constitute a
two-member family among heparin-binding growth factors.
The pattern of expression of HARP and its regulation suggest its
involvement in embryonic development. HARP is clearly expressed in
brain during the perinatal stage of rapid axon growth and synapse formation (3) and plays a role in bone formation (7). Even if HARP is
generally down-regulated after birth, it is also expressed during
adulthood and is important in cell growth and differentiation (reviewed
in Ref. 8). It seems particularly involved in tumor growth and
angiogenesis (9-11). HARP could also play a central role in normal
spermatogenesis, because knock out of the gene in mice may lead to
sterility in males (12).
HARP displays several biological activities in vitro.
Originally isolated as a neurite outgrowth-promoting protein (13), further studies have shown that this protein stimulated the cellular proliferation of a wide variety of cells, including fibroblast, epithelial, and endothelial cells (1, 9, 14, 15). In agreement with its
role in angiogenesis, HARP can induce migration of aortic bovine
endothelial cells in collagen (16) and enhances plasminogen activator
activity of the same cells (17). Until now, except for the neurite
outgrowth activity (18, 19), the cell surface molecules involved in
these biological activities are still poorly documented. However, it
seems that low and high affinity binding sites could be involved in its
mitogenic activity. The existence of high affinity receptors is
suggested by the phosphorylation of a 200-kDa protein in NIH-3T3 and
NB41A3 cells treated with HARP (20) and is strongly supported by the
ability of HARP to transduce a mitogenic signal through MAPK and
phosphatidylinositol 3-kinase pathways in BEL cells (21).
HARP is a 18-kDa protein that contains 24% of basic residues (18% of
lysines), mainly arranged in two clusters at the N- and C-terminal
regions, and five intrachain disulfide bonds, clearly demonstrated
using NMR experiments by the Rauvala group (22). The molecule is
organized in two Post- or cotranslational maturations of HARP have been considered as a
possible mechanism involved in HARP mitogenic activity. The cleavage
site of the signal peptide of HARP has been more particularly subject
to controversies between several groups. The N-terminal sequence of the
originally purified mature peptide from uterus or from conditioned
media of epithelial cells was GKKEKP corresponding to a 136-amino acid
protein (HARP136) (14, 25, 26). However, we have identified
a second cleavage site of the signal peptide leading to a 3-amino acid
extension AEA (HARP139), which seemed to be associated to
its mitogenic activity (16, 27). Similarly, discrepancies between
results of previous studies were observed for the putative C-terminal
maturation of HARP (21, 28).
To clarify the importance of N- and C-terminal maturations in
HARP mitogenic activity, C-terminally truncated mutants of HARP were
produced and purified, and the determinants playing a role in the
biological activity, including mitogenic, neurite outgrowth, and tumor
formation activities were investigated.
Materials--
Culture medium, fetal calf serum
(FCS), and G418 were supplied by Life Technologies (Cergy Pontoise,
France). Superblocker solution (Pierce) and horseradish
peroxidase-conjugated rabbit anti-goat and rabbit anti-mouse
immunoglobulins G (Jackson) were purchased from Interchim
(Montluçon, France), goat anti-human HARP antibodies from R&D
(Oxon, UK). Heparin-Sepharose gel and Mono-S column were from Amersham
Pharmacia Biotech (Orsay, France), Immobilon-P from Millipore Corp.
(Saint-Quentin en Yvelynes, France), BM chemiluminescence and Fugene6
from Roche Molecular Biochemicals Mannheim (Meylan, France) and
mouse anti-phospho-p44/p42 MAPK from New England BioLabs (Saint-Quentin
en Yvelynes, France). [methyl-3H]Thymidine was
provided by ICN (Orsay, France).
Mutagenesis--
The human HARP cDNA was subcloned
into the EcoRI site downstream of the cytomegalovirus
promoter of the eucaryotic expression plasmid pCDNA3 (Invitrogen,
Leek, Netherlands). The resulting plasmid, named pCDNA3-HARP, was
mutated using the QuickChange site-directed mutagenesis kit
(Stratagene, Saint-Quentin en Yvelynes, France).
Oligodeoxyribonucleotides were synthesized by Eurogentec (Belgium). The
presence of the mutations was confirmed by double-stranded DNA sequencing.
Cell Culture and Transfection--
NIH-3T3 cells expressing
c-Myc/6His-tagged HARP (clone HMH-C9) were maintained in Dulbecco's
modified Eagle's medium supplemented by 10% FCS and 400 µg/ml G418
(23). CHO-K1 cells (a generous gift from T. Melot, Institut Curie,
Paris) were cultured in Ham's F-12 supplemented by 10% FCS. For
transfection, 3 × 105 CHO-K1 cells per 60-mm dishes
were seeded in Ham's F-12 medium supplemented with 10% FCS and
transfected 24 h later with 4 µg of pCDNA3 or
pCDNA3-HARP plasmids using the liposomal system Fugene6 according
to the manufacturer's protocol. 48 h after transfection, cells
were selected for G418 resistance (800 µg/ml). Medium was changed
every 2 days until colonies formed. Each G418-resistant clonal
population was scrapped with a tip, resuspended, and cultured in the
selective medium. The clones secreting the largest amount of HARP were
selected. Several clones transfected with pCDNA3 were also selected
for control experiments (Mock cells).
Purification of HARP from Conditioned Media--
Tagged,
mutated, or wild type HARP proteins were purified as previously
described (16). Briefly, 8 × 106 NIH-3T3 or CHO-K1
cells were plated in 600-cm2 dishes and cultured for
72 h in complete medium. For CHO-K1 cells, the culture medium was
supplemented 24 h after seeding with 5 mM sodium
butyrate, which increased by 100-fold the expression level of
recombinant protein (29). Conditioned medium containing secreted HARP
proteins was buffered to pH 7.4 with 20 mM Hepes, ionic
strength adjusted to 0.5 M NaCl and then loaded onto a
10-ml heparin-Sepharose column. Bound proteins were eluted with 20 mM Hepes, 2 M NaCl, pH 7.4, and further
purified using a cation-exchange Mono-S column. The purification was
carried out in 50 mM Tris-HCl, pH 7.4, and proteins were
eluted using a 0.4 to 2 M NaCl gradient. Determination of
the N-terminal sequence was performed using the Protein Microsequencing
Service at the Pasteur Institute (Paris, France). The amount of HARP
proteins in each peak was evaluated using an immunometric assay
described in Soulié et
al.2
Thymidine Incorporation Assay--
3 × 104
NIH-3T3 wild type cells per well were seeded in 48-well plates for
24 h in DMEM supplemented by 10% FCS. Cells were then
serum-starved for 24 h, and samples were added. Cells were then
incubated at 37 °C and 7% CO2 for 18 h and
incubated further for 6 h with 0.5 µCi of
[methyl-3H]thymidine. Cells were then fixed
with 10% trichloroacetic acid, washed with water, and lysed with 0.1 N NaOH. Total incorporated radioactivity was counted using
a micro-beta scintillation counter (LKB, PerkinElmer Life Sciences,
Courtaboeuf, France). Similar protocol was used with BEL cells as
previously described (27).
Western Blotting Procedure--
Purified proteins were run on
SDS-15% polyacrylamide gel and electrotransferred to Immobilon-P
membrane in 10 mM CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid), pH 11, containing 10%
methanol. Nonspecific binding was prevented by incubating the membrane
for 20 min in the Superblocker solution at room temperature. The
membrane was then incubated overnight at 4 °C with goat anti-human
HARP antibodies (250 ng/ml) diluted in PBS containing 0.2% Tween 20 (PBS-T) and 3% Superblocker. After three washes with PBS-T, the
membrane was incubated 30 min at room temperature with the
peroxidase-conjugated anti-immunoglobulin goat antibodies diluted in
PBS-T, and the enzyme was detected using the BM chemiluminescence reagent.
Phosphorylation of MAPK--
2.5 × 105 NIH-3T3
cells were seeded in 35-mm culture dishes for 24 h, serum-starved
for 24 h, and stimulated for 5 min at 37 °C with samples. Cells
were lysed with electrophoresis sample buffer (50 mM
Tris-HCl, pH 6.8, 10% glycerol, 0.02% bromphenol blue, 2% SDS, and
5% Neurite Outgrowth Assays--
Cells from cerebral hemispheres of
18-day-old rat embryo were prepared as described previously (3). Brain
was dispersed into individual cells in DMEM containing 10% FCS, 6 mg/ml glucose, 100 units/ml penicillin, and 0.1 mg/ml streptomycin
using a 10-ml sterile syringe and centrifuged at 100 × g for 10 min. The pellet was suspended in the same medium
without FCS and containing 1 mg/ml bovine serum albumin. 2.5 × 104 cells were seeded in a 96-well EIA plate (Costar,
Brumath, France) precoated with 0.8, 1.6, or 3.2 µg/ml wild-type (wt)
HARP or mutant proteins. After 48 h, cells were stained with
May-Grünwald's solution and Giemsa stain. The number of neurites
per well was evaluated using phase contrast microscopy. Each experiment
was carried out in duplicate and representative results are shown.
Tumor Formation in Nude Mice--
Tumor formation in 5-week-old
female athymic nude mice (Nu/Nu, IFFA CREDO Laboratories) was tested by
subcutaneous injection of 106 cells suspended in 100 µl
of Ham's F-12 at a unique site. Tumor size was measured twice a week,
starting from the second week following injection. Mice were sacrificed
6 weeks after injection.
Contradictory results have been reported concerning the mitogenic
activity of HARP. Although only neurite outgrowth activity was
originally reported by Rauvala (13), further studies have suggested
that the mitogenic activity of HARP was dependent either on the
presence of three amino acids at the N terminus (16) or related to a
C-terminally truncated form (21). To clarify this point,
recombinant C-terminal-tagged molecules with the c-Myc/6His peptide,
which is useful to detect C-terminal processing, were produced from
NIH-3T3 cells (23). The recombinant proteins were purified,
characterized, and tested for their mitogenic activity.
N-terminal Processing of HARP and Mitogenic Activity
Purification of HARP from NIH-3T3 Cells--
Two liters of
conditioned medium were collected after a 72-h culture, and proteins
were purified using sequential heparin-Sepharose/Mono-S chromatographies, as described under "Experimental Procedures." Using a 0.4 to 2 M NaCl gradient for the elution of the
Mono-S column, five major fractions were collected (data not shown) and analyzed using SDS-polyacrylamide gel (Fig.
1A, lanes 1-5) and Western blot experiments (Fig. 1B). In fractions 2 and 4, two proteins with an apparent molecular mass of 21 kDa were mainly purified, in good agreement with the size of the C-terminal tag extension. Both polypeptides were immunodetected by the anti-HARP (Fig.
1B, lanes 2 and 4) and anti-c-Myc
(data not shown) antibodies. The fraction eluted with 0.61 M NaCl from the Mono-S column with an apparent molecular
weight of 21,000 (lane 2) corresponded to the
tagged-HARP139 molecule, whereas the fraction eluted with 0.67 M NaCl (lane 4) was determined to be
tagged-HARP136. Those two tagged-HARP molecules, therefore,
revealed N-terminal processing identical to what we previously
described (16). In fraction 4, two additional bands of 18 and 15 kDa
were slightly silver-stained, immunodetected with anti-HARP antibody
(Fig. 1B, lane 4), and could correspond to
C-terminal proteolytic cleavage, because they were not recognized by
the anti-c-Myc antibody (data not shown). Two 18-kDa proteins eluted
with 0.65 M NaCl (Fig. 1A, lane 3) and 0.69 M NaCl (Fig. 1A, lane 5)
were also purified and immunodetected with the anti-HARP antibody but
not using anti-c-Myc antibody (data not shown). They might correspond,
respectively, to HARP139 and HARP136 produced
endogenously by NIH-3T3 cells, but we cannot exclude a cleavage of the
c-Myc/6His epitope, even if the purification was carried out in the
presence of protease inhibitors. During this experiment, two proteins
with apparent molecular masses of 65 and 30 kDa were isolated in
fraction 1 (arrows in Fig. 1A, lane
1). N-terminal sequencing and Western blot experiments with an
anti-HGF
The mitogenic activity of the recombinant HARP isolated as described
above was then assayed on serum-starved NIH-3T3 cells (Fig.
2A). Aliquots of each
fractions, as mentioned in the figure legend, were added to the cells,
and mitogenic activity was determined as described under
"Experimental Procedures." As shown in Fig. 2A, the
fractions containing the HARP139 (fraction 2, tagged-HARP139; fraction 4, HARP139) as well as
HARP136 form (fraction 3, tagged-HARP136; fraction 5, HARP136) induced cell proliferation in a
dose-dependent manner. A 4-fold increase of tritiated
thymidine incorporation in NIH-3T3 cells as compared with the control
was observed for the highest concentration of each fraction and was
similar to what was obtained with 10% FCS (Fig. 2A).
Specific activities were calculated by estimating the concentration of
HARP in the different fractions using an immunometric assay that we
have developped.2 The ED50 level for
HARP139 was higher than for HARP136,
i.e. 2 and 6 nM, respectively. Similar results
were obtained using BEL cells (data not shown). However, although HGF
was only immunodetected in the first eluted fraction from Mono-S
chromatography, it was important to rule out the possibility of a
cross-contamination by HGF in other eluted fractions containing HARP.
In this respect, NIH-3T3 cells were stimulated with a saturating dose
of HGF in the presence or not of the different HARP fractions described above. As shown in Fig. 2B, addition of an aliquot of each
purified HARP fraction induced a higher stimulation than those achieved by HGF alone. The same overstimulation of DNA synthesis was observed for FGF-2 used in this experiment as positive control. These results clearly indicated that the effects of HGF and HARP on DNA synthesis were independent and suggested that the stimulation observed in fractions 2-5 was not due to HGF contaminations. However, considering the overstimulation obtained with fraction 2, which was weaker than
expected, a slight contamination of this fraction by HGF cannot be
completely ruled out.
Production of Recombinant HARP from CHO-K1 Cells--
The
N-terminal maturation of HARP was also studied using a CHO-K1 cell
expression system. After transfection with the cDNA encoding HARP
protein, CHO-K1 clonal populations overexpressing HARP were selected
using G418, and HARP was purified from the conditioned medium as
described under "Experimental Procedures." Under these conditions,
only HARP136 was isolated. Neither HARP139 nor
HGF proteins have been identified. HARP136 isolated from
CHO-K1 cells displayed mitogenic activity for NIH-3T3 cells (see below) and BEL cells (not shown). However, HARP136 produced by
CHO-K1 cells was about three times less active than HARP136
produced in NIH-3T3 cells (ED50 of 20 nM
versus 6 nM). As control, purification steps
were carried out from the conditioned medium of CHO-K1 cells transfected with vector alone, and no mitogenic activity was yielded in
the collected fractions from the Mono-S chromatography (not shown).
Involvement of C-terminal Determinants for the Mitogenic Activity
of HARP
As mentioned in the introduction, truncated forms of HARP at the
C-terminal region have been identified in SW13 transfected with HARP
cDNA and in Swiss 3T3 cells (21, 28). Involvement of this
C-terminal maturation in HARP mitogenic activity has not been clearly
established, and, to further investigate this point, C-terminal
truncated HARP proteins were produced in CHO-K1 cells.
Two different C-terminal truncated mutants were constructed by adding a
stop codon (Fig. 3A): (i)
H-sheet
domains, each domain containing a cluster of basic residues. To assess
determinants involved in the biological activities of HARP,
C-terminally truncated proteins were produced in Chinese hamster
ovary-K1 cells and tested for their mitogenic, tumor formation in nude
mice and neurite outgrowth activities. Our data clearly indicate that
the residues 111-136 of the lysine-rich C-terminal domain are involved
in the mitogenic and tumor formation activities of HARP. Correlatively, no signal transduction was detected using the corresponding mutant, suggesting the absence of HARP binding to its high affinity receptor. However, this C-terminal domain of HARP is not involved in the neurite
outgrowth activity. We also demonstrate that HARP signal peptide
cleavage could led to two maturated forms that are both but
differentially mitogenic.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet domains linked by a flexible linker, and
each of these two domains includes one heparin-binding site. At least
one heparin-binding site is involved in the dimerization of this growth
factor (23) and is important for HARP mitogenic activity, because this
activity on BEL cells is modulated by exogenous addition of
glycosaminoglycans (24). Furthermore, treatment of BEL cells with
heparinase III abolished HARP mitogenic activity, which could be
restored by the addition of soluble heparin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol), and the presence of phospho-p42/p44 MAPK was
detected by Western blot using the procedure described above except
that proteins were run on SDS-10% polyacrylamide gel,
electrotransferred in 25 mM Tris, pH 8.3, containing 200 mM glycine, and 10% ethanol, and Immobilon-P membrane
incubated in PBS-T supplemented with 2% (w/v) powdered milk. The
monoclonal anti-phospho-p42/p44 antibodies was diluted to obtain a 1 µg/ml concentration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
antibody (inset of Fig. 1B)
identified them to be the
and
chains of the hepatocyte growth
factor. We can also note that bovine serum albumin was identified in
fraction 2 (Fig. 1A, lane 2).
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Fig. 1.
Purification of recombinant HARP from NIH-3T3
cells. Recombinant HARP was purified from conditioned medium of
NIH-3T3 cells expressing HARP tagged with the c-Myc/6His epitope, using
sequential heparin-Sepharose and Mono-S chromatographies. A,
20-µl aliquots of each fraction (lanes 1-5), collected
from the Mono-S column using a 0.4 to 2 M NaCl gradient,
were electrophoresed using a 15% SDS-PAGE, and proteins were detected
using silver staining. HGF and HGF
correspond to the two subunits of this growth factor. B,
Western blot of 5 µl of each fraction using an anti-human HARP
antibody after 15% SDS-PAGE electrophoresis and transferred to
Immobilon-P membrane. Inset, similar experiment to
B but using an anti-HGF
antibody.
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Fig. 2.
Mitogenic activity of recombinant HARP
produced from NIH-3T3 cells. A, stimulation of
[3H]thymidine incorporation of serum-starved NIH-3T3
cells during 18 h by 0.1-, 1-, and 10-µl aliquots of HARP
fractions obtained from the Mono-S chromatography described in Fig. 1.
FGF-2 and FCS are used as positive controls. B,
overstimulation experiments. Serum-starved NIH-3T3 cells were
stimulated by a combination of a saturating dose of HGF (10 µl of
fraction 1) and 1 µl of each mono-S HARP fraction (described in Fig.
1). Purified recombinant FGF-2 (10 ng/ml) was used as a positive
control of overstimulation. The results are the means of three separate
experiments carried out in triplicate and the standard errors are
indicated. For more details concerning the thymidine incorporation, see
"Experimental Procedures."
129-136 in which the last 8 amino acids of HARP were suppressed
but the C-terminal cluster of lysines was conserved and (ii)
H
111-136 in which the consensus sequence, possibly involved in
angiogenic activity (30) and containing the cluster of lysines, was
deleted. However, all the cysteines involved in disulfide bridges were
conserved. As described above, expression plasmids coding for the
mutant proteins were transfected in CHO-K1 cells, selected using G418
and the HARP expression levels of each stably transfected cell line
were determined by HARP immunometric assay2 (data not
shown). HARP proteins were purified from 0.4 liter of transfected
CHO-K1 conditioned media according to the procedure described above,
and isolated proteins were analyzed by Western blotting experiment with
an anti-HARP antibody (Fig. 3B). For each mutant proteins,
the molecular weight observed was in agreement with the size of the
deletion (17,200 and 14,500 for H
129-136 and H
111-136,
respectively).
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Fig. 3.
Mitogenic activity of wild type and mutant
HARP produced from CHO-K1 cells. A, the full-length
cDNA encoding HARP includes a signal peptide (hatched
box) and two lysine-rich clusters (black box).
H 129-136, a TAG stop codon, was introduced at position 129 (K,
AAG); H
111-136, a TAA stop codon was introduced at position 111 (K,
AAA). Wt HARP cDNA and the mutant forms were transfected in CHO-K1
cells. G418-resistant clones were selected as described under
"Experimental Procedures." B, wt HARP or mutant proteins
were purified from conditioned medium of each clone as described in
Fig. 1, run on a 15% SDS-PAGE, electrotransferred to Immobilon-P
membrane, and immunoblotted with an anti-HARP antibody (wt HARP,
lane 1; H
111-136, lane 2; H
129-136,
lane 3). C, stimulation of
[3H]thymidine incorporation in serum-starved NIH-3T3
cells treated with different concentrations of purified wt or mutant
HARP proteins. [3H]Thymidine incorporation is expressed
as compared with the negative control values (cells treated with
buffer).
Mitogenic Activity of C-terminal Truncated HARP
Proteins--
Mitogenic activity of the C-terminal truncated HARP
proteins was tested on serum-starved NIH-3T3 cells. A dose-response
curve was performed for mutant proteins as well as wt HARP (Fig.
3C). Both wt HARP and H129-136 mutant proteins presented
similar mitogenic activities with an ED50
20 nM, whereas H
111-136 mutant protein was not mitogenic.
The mitogenic activity profile was identical for BEL cells (data not
shown), and a similar result was obtained with another H
111-136
expressing clone. HARP mitogenic activity had been associated to
tyrosine kinase activation and involvement of MAPK and
phosphatidylinositol 3-kinase pathways in BEL cells (21). The ability
of wt HARP and H
111-136 mutant protein to induce MAPK
phosphorylation was therefore tested on NIH-3T3 cells (Fig.
4). As observed in BEL cells (21),
stimulation of NIH-3T3 cells with wt HARP led in a
dose-dependent manner to the phosphorylation of ERK1 and
ERK2 (Fig. 4, lanes 3 and 4). However, as
expected from cell stimulation experiments, no phosphorylation was
observed with the H
111-136 mutant (Fig. 4, lanes 5 and
6). Amino acids 111-136, therefore, seemed to play a key
role in the mitogenic activity of HARP, and we then decided to compare
the H
111-136 mutant and the wt HARP protein upon different
biological activities known for HARP.
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Tumor Formation in Nude Mice--
HARP had been previously
described to induce tumor formation in nude mice (31). Hence, 1 × 106 CHO-K1 cells expressing wt HARP or H111-136 mutant
were injected subcutaneously in nude mice. pCDNA3-transfected or
parental cells were used as controls. Two weeks after the injection,
two mice injected with cells expressing wt HARP had developed tumors
and four mice (out of seven) had tumors 4 weeks later (Table
I). No mice injected with parental cells
or H
111-136-expressing cells had tumors, and only one injected with
pCDNA3-transfected cells started to develop a tiny tumor after 6 weeks. To verify that tumors had derived from injected cells, tumor
fractions were dispersed in culture medium and cultured in the presence
of 800 µg/ml G418. Under these conditions, most of the cells from the
tumors appeared to be G418-resistant and expressed HARP as demonstrated
by immunohistochemistry experiments (data not shown). Accordingly, the
C-terminal 26-amino acid tail is also involved in tumor formation.
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Neurite Outgrowth Activity--
In contrast to the controversial
reports concerning the mitogenic activity of the recombinant HARP,
which seemed to depend on the system used (reviewed in Ref. 8), each
purified protein reported until now displayed a neurite outgrowth
activity. These data suggested that mitogenic and neurite
outgrowth-promoting activities were mediated through different
pathways. The neurite outgrowth-promoting activity of wt HARP and
H111-136 (lacking mitogenic activity and tumorigenicity) produced
in CHO-K1 cells was therefore evaluated. Neurons were seeded on a plate
previously coated with different concentrations of H
111-136 or wt
HARP (0.8, 1.6, or 3.6 µg/ml). Forty hours later, the neurite
outgrowth-promoting activity was determined by counting the number of
neurite cells per well using phase contrast microscopy. Both
mutant (Fig. 5, B and
D) and wild type (Fig. 5C) proteins were able to
induce neurite outgrowth, in a dose-dependent manner (Fig.
5E). Both forms were active at 0.8 µg/ml, and a 6-fold
increase of the number of neurites, as compared with the negative
control performed without protein, was observed for the highest
concentration (3.6 µg/ml) and for 20 µg/ml polylysine as well (not
shown). From this experiment, we can conclude that the C-terminal 26 amino acids are not involved in the neurite outgrowth activity of
HARP.
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DISCUSSION |
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Since the description of HARP as a mitogenic factor in 1989 (25), the essential objective of several groups, including ours, has been the molecular identification of cell surface macromolecule(s) linked to this activity. Until now, no candidate has been characterized, and, in this context, structure-function studies of HARP have been considered as a major development in the understanding of the mechanism involved in its mitogenic activity.
In previous reports, we described the existence of two different
processing sites of the signal peptide for recombinant proteins from
HARP cDNA-transfected NIH-3T3 cells (16) and for native HARP
expressed by BEL cells (27) leading to the production of two molecular
forms, HARP136 and HARP139, which differ by an
extension of 3 amino acids at the N-terminal part of the molecule. To
confirm these results, recombinant HARP was produced by two different cell lines, including NIH-3T3 cells and CHO-K1 cells. As expected, both
HARP136 and HARP139 proteins were expressed by
NIH-3T3 cells. However, only HARP136 was detected in
conditioned medium of CHO-K1 cells. The absence of HARP139
had also been observed in the conditioned medium from MDA-MB231 cells
(26) that endogenously produced HARP and from SW13 transfected with
HARP cDNA (14). The cleavage of the signal peptide seems to be
dependent on the cells that produce HARP and could explain the
discrepancy between the results related to the existence of
HARP139 obtained by various laboratories in different
tissues or cell lines. In contrast to our previous studies (9), both
molecular forms of HARP were mitogenic for NIH-3T3 and BEL cells,
demonstrating that the presence of the 3 amino acids AEA at the N part
of the molecule cannot be considered as the main determinant for HARP
mitogenic activity. This suggestion is strongly supported by the
production of HARP1391-3 mutant protein expressed in
CHO-K1 cells, which displayed a mitogenic activity for NIH-3T3
cells.3 However, we showed
that the different processing of HARP, yielding the HARP136
or HARP139, could be a possible mechanism involved in the
modulation of the mitogenic activity of this growth factor. This
possibility was suggested by the difference between specific activities
registered for each molecular form of HARP. The absence of mitogenic
activity for HARP136 that we have previously described could be due to structural or/and post-translational modifications, which could be different from one clone to another or depend on the
mammalian expression system used. This last hypothesis is reinforced by
the difference in the specific activity of HARP136 observed
with NIH-3T3 and CHO-K1 cells. Indeed, the value of ED50 of
HARP136 is about three times higher when the protein is
purified from NIH-3T3 cells as compared with CHO-K1 cells.
In contrast to a previous report, proposing that the mitogenic
form of HARP was a C-terminally truncated form of 14-kDa HARP (21), our
results demonstrated that the C-terminal part of the molecule is
clearly involved in the mitogenic activity of HARP and, consequently,
that the full-length protein is mitogenic. This assumption is also
supported by the fact that fractions corresponding to c-Myc/6His-tagged
molecules (see Fig. 1), further purified using an Ni2+-trap
column, were still active (data not shown). The amino acids 111-128
seem to play a key role in the mitogenic activity of this growth
factor, because the H111-136 mutant displays no mitogenic activity
when the activity of H
129-136 mutant is similar to the wild type
form. In addition, tumor formation in nude mice was no longer observed
with H
111-136 protein, suggesting that a similar domain was
involved in both mitogenic and tumor formation potency. Our results, in
addition to a recent report showing that H
122-136 induced tumor
formation (32), suggest that the 111-122 domain KLTKPKPQAESK is
involved at least in the tumor formation. It is also clear from our
data that determinants involved in HARP mitogenic and neurite outgrowth
activities are different. Therefore, the nonmitogenic H
111-136
mutant is still able to induce differentiation of primary cultures of
neurons, suggesting that the 111-136 domain of HARP, including the
C-terminal cluster of lysines, is not involved in neurite outgrowth activity.
The correct folding of mutant proteins is a complex problem of
mutagenesis experiments, but recent structural data suggest that
folding of the HARP mutants described here could be very similar to the
wt protein (22). The Rauvala group reported that HARP is organized as
two -sheets domains connected by a flexible linker. In H
111-136
mutant proteins, the absence of these two structural domains is
unlikely, for the following reasons: (i) All the cysteines known to be
involved in disulfide bridges (33, 34) are conserved and may confer a
tridimensional structure closed to the wild type protein; (ii) The
C-terminal part of the molecule that we have deleted appeared to form
random coils (22); (iii) HARP
111-136 is still recognized by a
polyclonal antibody raised against wt HARP in the immunometric
assay2; (iv) HARP
111-136 is able to induce a neurite
outgrowth (see above).
Despite the lack of a detectable secondary structure in NMR and CD
experiments (22), the C-terminal part of this molecule appears to be
very important for the mitogenic activity and could be involved in the
binding to high affinity receptors. This hypothesis was supported by
signal transduction studies, because stimulation of the MAPK pathway,
known to be involved in HARP mitogenic activity in BEL cells (21), was
abolished for H111-136.
Taken together, these results suggest that multiple cell-specific
receptors could then mediate diverse biological activities. A more
complex pattern could be considered if the cell surface molecule
involved in biological activities of HARP depends on the cells or tissue.
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FOOTNOTES |
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* This work was supported in part by grants from Association pour la Recherche sur le Cancer (number 5257), Ministère de l'Eduction Nationale (DRED) and CNRS.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors equally contributed to this work.
§ Recipient of a grant from the Ministère de la Recherche et de l'Enseignement Supérieur.
¶ To whom correspondence should be addressed: Tel.: 33-1-45-17-17-97; Fax: 33-1-45-17-18-16; E-mail: Courty@univ-paris12.fr.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M010913200
2 P. Soulié, M. Héroult, I. Bernard-Pierrot, M. E. Kerros, P. E. Milhiet, J. Delbé, D. Barritault, D. Caruelle, and J. Courty, manuscript submitted.
3 I. Bernard-Pierrot, J. Courty, J. Delbé, and P. E. Milhiet, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HARP, heparin affin regulatory peptide; BEL, bovine epithelial lens cells; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FGF-2, basic fibroblast growth factor; HGF, hepatocyte growth factor; MAPK, mitogen-activated protein kinase; PBS-T, PBS containing 0,2% Tween 20; wt, wild type; CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid; ERK1/2, extracellular signal-regulated kinases 1 and 2.
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