The glycosaminoglycan-binding domain of PRELP acts as a cell type-specific NF-kappaB inhibitor that impairs osteoclastogenesis.

Abstract

Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a glycosaminoglycan (GAG)- and collagen-binding anchor protein highly expressed in cartilage, basement membranes, and developing bone. We observed that PRELP inhibited in vitro and in vivo mouse osteoclastogenesis through its GAG-binding domain ((hbd)PRELP), involving (a) cell internalization through a chondroitin sulfate- and annexin II-dependent mechanism, (b) nuclear translocation, (c) interaction with p65 nuclear factor kappaB (NF-kappaB) and inhibition of its DNA binding, and (d) impairment of NF-kappaB transcriptional activity and reduction of osteoclast-specific gene expression. (hbd)PRELP does not disrupt the mitogen-activated protein kinase signaling nor does it impair cell survival. (hbd)PRELP activity is cell type specific, given that it is internalized by the RAW264.7 osteoclast-like cell line but fails to affect calvarial osteoblasts, bone marrow macrophages, and epithelial cell lines. In vivo, (hbd)PRELP reduces osteoclast number and activity in ovariectomized mice, underlying its physiological and/or pathological importance in skeletal remodeling.

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JCB: Article
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 187 No. 5 669–683
www.jcb.org/cgi/doi/10.1083/jcb.200906014
JCB669
N. Rucci, A. Rufo, and M. Alamanou contributed equally to this paper.
Correspondence to Anna Teti: annamaria.teti@univaq.it
Abbreviations used in this paper: ALP, alkaline phosphatase; CTX, C-terminal
collagen I cross-links; GAG, glycosaminoglycan; LRR, leucine-rich repeat;
M-CSF, macrophage colony-stimulating factor; NF-B, nuclear factor B; PRELP,
proline/arginine-rich end LRR protein; RANK, receptor activator of NF-B;
RANKL, RANK ligand; RIPA, radioimmunoprecipitation assay; SLRRP, small LRR
protein; TRAcP, tartrate-resistant acid phosphatase.
Introduction
The proline/arginine-rich end leucine-rich repeat (LRR) protein
(PRELP) is a 58-kD heparin/heparan sulfate–binding protein
first discovered in articular cartilage but present also in several
connective tissue extracellular matrices. The protein comprises
382 aa residues, including a 20-residue signal peptide. It be-
longs to a subfamily of LRR proteins in the extracellular matrix.
Members encompass several small LRR proteins (SLRRPs), in-
cluding the chondroitin/dermatan sulfate proteoglycans decorin
and biglycan and the keratan sulfate proteoglycans fibromodulin
and lumican (Iozzo and Murdoch, 1996). 10–11 adjacent LRRs
characterize this subfamily, flanked at either end by disulphide-
bonded domains (Heinegård et al., 2002).
N-linked oligosaccharides are present in the central LRR
domain of PRELP (Bengtsson et al., 1995), whose name reflects
the abundance of proline and arginine in its N-terminal domain
(Bengtsson et al., 1995). Compared with many of the other
members of the SLRRP subfamily, PRELP has two atypical
features. First, it does not contain glycosaminoglycan (GAG)
chains; second, the N-terminal region, which is unique and con-
served between rodents, bovine, and humans, binds heparin and
heparan sulfate (Bengtsson et al., 2000). N-terminally truncated
PRELP lacking this region cannot bind heparin, whereas a 6-mer
heparin oligosaccharide is the smallest size showing some affin-
ity to PRELP. Binding increases with length up to 18-mer and
was found to depend on the degree of sulfation of heparin and
heparan sulfate (Bengtsson et al., 2000). The protein binds
collagens I and II with high affinity (Bengtsson et al., 2002) via
its LRR domain, whereas the N-terminal part of PRELP can
bind the heparan sulfate of perlecan or bind fibroblasts via
surface heparan sulfate proteoglycans (Bengtsson, 1999), thus
P
tilage, basement membranes, and developing bone. We
observed that PRELP inhibited in vitro and in vivo mouse
osteoclastogenesis through its GAG-binding domain
(hbdPRELP), involving (a) cell internalization through a chon-
droitin sulfate– and annexin II–dependent mechanism,
(b) nuclear translocation, (c) interaction with p65 nuclear
factor B (NF-B) and inhibition of its DNA binding,
and (d) impairment of NF-B transcriptional activity and
roline/arginine-rich end leucine-rich repeat protein
(PRELP) is a glycosaminoglycan (GAG)- and collagen-
binding anchor protein highly expressed in car-
reduction of osteoclast-specific gene expression. hbdPRELP
does not disrupt the mitogen-activated protein kinase sig-
naling nor does it impair cell survival. hbdPRELP activity is
cell type specific, given that it is internalized by the
RAW264.7 osteoclast-like cell line but fails to affect
calvarial osteoblasts, bone marrow macrophages, and
epithelial cell lines. In vivo, hbdPRELP reduces osteoclast
number and activity in ovariectomized mice, underlying
its physiological and/or pathological importance in skel-
etal remodeling.
The glycosaminoglycan-binding domain of PRELP
acts as a cell type–specific NF-B inhibitor that
impairs osteoclastogenesis
Nadia Rucci,1 Anna Rufo,1 Marina Alamanou,1 Mattia Capulli,1 Andrea Del Fattore,1 Emma Åhrman,2
Daria Capece,1 Valeria Iansante,1 Francesca Zazzeroni,1 Edoardo Alesse,1 Dick Heinegård,2 and Anna Teti1
1Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy
2Section for Rheumatology, Department of Clinical Sciences, Lund University, SE-22184 Lund, Sweden
© 2009 Rucci et al. This article is distributed under the terms of an Attribution–
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tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y

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JCB • VOLUME 187 • NUMBER 5 • 2009 670
with vehicle, hbdPRELP, or the control peptide and with the
bisphosphonate alendronate as a reference drug. hbdPRELP sig-
nificantly reduced the ovariectomy-induced increase of the urine
bone resorption marker C-terminal collagen I cross-links (CTX;
Fig. 2 A). In histological sections of proximal tibia, secondary
spongiosa osteoclast number as well as osteoclast surface per
bone surface area were decreased in ovariectomized mice treated
with hbdPRELP versus vehicle- and control peptide–treated mice
(Fig. 2, B–D). Consistently, the analysis of trabecular bone struc-
tural parameters (Fig. 2, E–H) showed the ability of hbdPRELP
to significantly reduce the ovariectomy-induced bone loss.
Mechanism of action
We next sought to establish when the peptide was active during
in vitro osteoclast formation. Fig. 3 A shows that hbdPRELP re-
duced osteoclastogenesis both when given for the entire period
of culture and during the last 3 d, whereas it was inactive when
administered only throughout the first 3 d. The effect was irre-
versible in that treatment with 15 µM hbdPRELP for 5 d followed
by hbdPRELP withdrawal, and continuation of the culture for a
further 5 d did not result in the induction of osteoclastogenesis
(unpublished data). Moreover, hbdPRELP significantly reduced
adhesion when cells were pretreated in suspension with the
peptide before seeding them in the culture dish (Fig. 3 B). This
result suggests interference with cell surface molecules, per-
haps proteoglycans.
Role of cell surface proteoglycans
To address whether cell surface proteoglycans carrying heparan
sulfate chains were involved in the inhibitory effect by hbdPRELP
on osteoclast formation, we performed osteoclastogenesis as-
says in the presence of heparinase III. Surprisingly, this treat-
ment was unable to rescue osteoclast formation in the presence
of hbdPRELP (Fig. 3 C). Therefore, we next investigated the in-
volvement of cell surface proteoglycans substituted with chon-
droitin sulfate chains (Deepa et al., 2004). Indeed, we observed
that pretreatment of prefusion osteoclast cultures with chon-
droitinase ABC prevented hbdPRELP from inhibiting osteoclast
formation (Fig. 3 D). Confocal microscopy with a specific anti-
body confirmed that chondroitin sulfate was expressed by pre-
fusion osteoclasts and was removed from the cell surface after
treatment with chondroitinase (Fig. 3 E). Moreover, chondroitin
sulfate largely colocalized with a hbdPRELP peptide tagged with
biotin (BiotinhbdPRELP; Fig. 3 F).
Internalization of hbdPRELP
The hbdPRELP sequence contains eight arginine residues in
motifs typical of viral proteins prone to be internalized by eukary-
otic cells (Futaki et al., 2001). Therefore, we hypothesized that
hbdPRELP may be internalized and affect osteoclastogenesis via
an intracellular target. To address this aspect, we vitally incu-
bated prefusion osteoclasts with BiotinhbdPRELP and monitored
its uptake by confocal microscopy. After 5 min, BiotinhbdPRELP
was mostly localized at the cell surface in structures reminis-
cent of membrane rafts (Fig. 4 A, a), whereas after 10 min, it
was internalized in endosomal vesicles (Fig. 4 A, b). Interest-
ingly, after 20 min, BiotinhbdPRELP was mainly found in the
serving as a linker between these proteoglycans and the extra-
cellular matrix.
The gene encoding PRELP maps at chromosome 1q32,
and PRELP mRNA transcripts were found in articular chondro-
cytes, osteoblasts, and osteosarcoma cells of various species
(Bengtsson et al., 2000). The protein was also found at base-
ment membranes of skin, testis, and Bowman’s capsule of the
kidney (Bengtsson et al., 2002). PRELP plays a role in eye and
skin (Reardon et al., 2000; Grover et al., 2007). The protein is
highly expressed in human sclera, and mutations have been
found in advanced myopia (Majava et al., 2007). PRELP muta-
tions are also involved in the pathogenesis of Hutchinson-Gilford
progeria (Lewis, 2003), which is characterized, among other
symptoms, by scleroderma, achondrogenesis, bone deformities,
and osteoporosis (Hennekam, 2006).
Although PRELP was found in the skeleton expressed by
chondrocytes and osteoblasts, there is no direct information re-
garding the role of the protein in skeletal remodeling. We sought
to identify its role in bone homeostasis using an N-terminal pep-
tide corresponding to the entire heparin-binding domain of PRELP
(hbdPRELP). The peptide was tested in in vitro cultures of mouse
osteoblasts and osteoclasts and in a mouse model of bone loss. Al-
though hbdPRELP had no effect on osteoblasts and other cell types,
it impaired osteoclastogenesis and bone resorption by a mecha-
nism requiring its internalization, translocation to the nucleus, and
inhibition of the transcription factor nuclear factor B (NF-B).
Results
Effect of hbdPRELP on osteoclastogenesis
and bone resorption
In vitro osteoclastogenesis assays showed that hbdPRELP but not
our control heparin-binding peptide remarkably reduced osteo-
clast formation from unfractionated bone marrow cells treated
with 1,25(OH)2VitaminD3 (Fig. 1 A). The hbdPRELP effect was
concentration dependent, with a calculated IC50 of 7.3 µM and a
narrow range of optimal concentrations (Fig. 1 B). Consistent
with the impairment of osteoclast generation, hbdPRELP signifi-
cantly reduced pit number (Fig. 1 C). Furthermore, hbdPRELP
appeared to have a direct effect on the osteoclast lineage,
as demonstrated by the inhibition of osteoclastogenesis in puri-
fied bone marrow macrophage cultures treated with macrophage
colony-stimulating factor (M-CSF) and receptor activator of
NF-B (RANK) ligand (RANKL; Fig. 1 D). Notably, this effect
was observed also using the intact PRELP protein, which showed
a potency similar to that of the hbdPRELP peptide (Fig. 1 E). Fi-
nally, in osteoclasts previously differentiated by treatment with
M-CSF and RANKL and then transferred onto bone slices and
subsequently treated with hbdPRELP for 48 h, the peptide failed to
affect pit formation, suggesting that the major effect is exerted on
the mechanism of osteoclast formation rather than on that of bone
resorption (Fig. S1 A).
Effect of hbdPRELP in vivo
To assess whether hbdPRELP may also have a role in vivo, we
increased osteoclast activity and induced bone loss in female
mice by ovariectomy (Idris et al., 2008) and treated the animals

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671PRELP and osteoclastogenesis • Rucci et al.
Figure 1. Effect of hbdPRELP on osteoclastogenesis and bone resorption. (A and B) Unfractionated mouse bone marrow cells were incubated in the presence
of 1,25(OH)2VitaminD3 with vehicle, 15 µM hbdPRELP, or 15 µM of control peptide (A) or with the indicated concentrations of hbdPRELP (B). Osteoclasts
were then stained for TRAcP, enumerated, and expressed as the percentage of vehicle treated. (C) Pit index of cells cultured as in A but onto bone slices.
(D) Bone marrow macrophages were incubated for 6 d with 50 ng/ml M-CSF and 120 ng/ml RANKL plus vehicle or 15 µM hbdPRELP. Osteoclasts were
then assessed by TRAcP staining (top) and enumerated (bottom). (E) Bone marrow macrophages were treated with 15 µM of control peptide, 15 µM
hbdPRELP, or 15 µM of intact PRELP, and osteoclastogenesis was assessed as described in A. (A–E) Results are the mean ± SEM of three independent
experiments (*, P < 0.01).

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JCB • VOLUME 187 • NUMBER 5 • 2009 672
Figure 2. Effect of hbdPRELP in vivo. Ovariectomized (OVX) mice were treated with vehicle, 10 mg/kg body weight of hbdPRELP, or control peptide and
1 mg/kg body weight of alendronate as reference drug 5 d/wk for 5 wk. (A) Quantification of the bone resorption marker CTX in urine samples. (B) Tibial
sections stained for TRAcP activity (purple stain) to evidence osteoclasts. (C–H) Measurement of osteoclast number/bone surface (OcN/BS; C), osteoclast
surface/bone surface (OcS/BS; D), bone volume/tissue volume (BV/TV; E), trabecular number (Tb N; F), trabecular thickness (Tb Th; G), and trabecular
separation (Tb Sp; H). (A and C–H) Data are the mean ± SD of five mice per group (*, P < 0.05 vs. sham; #, P < 0.05 vs. control peptide and vehicle;
and +, P < 0.05 vs. control peptide).

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673PRELP and osteoclastogenesis • Rucci et al.
Figure 3. Role of cell surface proteoglycans in hbdPRELP-induced inhibition of osteoclastogenesis. (A) Purified mouse bone marrow macrophages, incubated
with M-CSF and RANKL, were treated with vehicle (0) or 15 µM hbdPRELP for the entire time frame of the cultures (1–6 d), for the first 3 d (1–3), or for the
last 3 d (4–6). (B) Osteoclast cultures, differentiated by M-CSF and RANKL, were trypsinized. Cells were then pretreated in suspension with vehicle, 15 µM
hbdPRELP, or 15 µM of control peptide and allowed to adhere to substrate for the times indicated. Attached cells were stained for TRAcP (top) and enumer-
ated (bottom). (C and D) Prefusion osteoclasts were pretreated with 2 U/ml heparinase III (C) or 0.45 U/ml chondroitinase ABC (D) before the addition
of hbdPRELP, and then multinucleated osteoclasts were stained for TRAcP and enumerated. (E) Prefusion osteoclasts were pretreated with vehicle or with
0.45 U/ml chondroitinase ABC, fixed, and incubated with an anti–chondroitin sulfate antibody (green), with no permeabilization to assess the content of
cell surface chondroitin sulfate. Cells were also stained with DAPI (blue) to detect nuclei. Pictures are the results of the merge between the two fluorescences.
(F) Prefusion osteoclasts were treated with a biotin-tagged hbdPRELP (BhbdPRELP), fixed, permeabilized, and incubated with an anti–chondroitin sulfate anti-
body to detect colocalization of tagged hbdPRELP with cell surface and vesicular chondroitin sulfate (biotin 594-streptavidin [streptav] was used as a negative
control). (A–D) Results are representative or the mean ± SEM of three independent experiments (*, P < 0.001).

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JCB • VOLUME 187 • NUMBER 5 • 2009 674
nucleus, and several endosomal vesicles appeared in the pro-
cess of BiotinhbdPRELP transfer to the nuclear compartment
(Fig. 4 A, c). Mature multinucleated osteoclasts were also able
to internalize BiotinhbdPRELP and retained it in the nuclei after
20 min of vital incubation (Fig. 4 A, d). Control cultures incu-
bated with biotin-594 streptavidin alone were negative (Fig. 4 A, e).
Similar internalization was observed with another tagged pep-
tide, Alexa Fluor 488–hbdPRELP (Fig. S1 B).
BiotinhbdPRELP internalization required active cellular
processes given that the events observed at 37°C (Fig. 4 A, f)
were inhibited when the cells were incubated at 4°C (Fig. 4 A, g).
Furthermore, pretreatment with chondroitinase ABC abol-
ished BiotinhbdPRELP membrane binding and internalization
(Fig. 4 A, h). Finally, we fixed and permeabilized prefusion
osteoclasts and incubated these cells with Alexa Fluor 488–
hbdPRELP. Indeed, we observed that fluorescent hbdPRELP
not only decorated membrane and intracellular compartments
but was also largely localized in the nuclei (Fig. 4 A, i).
Interaction with cellular proteins
To address which candidate protein could interact with Biotinhbd
PRELP, we performed SDS-PAGE and blot transfer of untreated
prefusion osteoclast total cell lysates, incubated the blots with
BiotinhbdPRELP, and visualized the biotin by HRP-conjugated
streptavidin and ECL. Indeed, several specific bands were re-
vealed, among which, those most evident had apparent molecu-
lar masses of 35 and 65 kD (Fig. 4 B).
One important 35-kD protein known to have a central role
in endocytosis is annexin II (Gerke et al., 2005). Indeed, by
stripping the membrane and reprobing it with an annexin II
antibody, we identified this protein at 35 kD (Fig. 4, compare B
[right] with C [left]). CD44 represents another cell surface pro-
tein known to have roles in endocytosis in many cell types
(Aguiar et al., 1999; Jiang et al., 2002) and in the fusion process
of osteoclast precursors (Kania et al., 1997; Sterling et al., 1998;
Suzuki et al., 2002). However, reprobing the membrane with an
antibody recognizing CD44 revealed a band at 80 kD (Fig. 4 C,
middle), which did not correspond to any of the specific Biotin-
hbdPRELP bands observed in Fig. 4 B.
NF-B is a transcription factor crucial to osteoclast devel-
opment and function. One of the components labeled by Biotin-
hbdPRELP in Fig. 4 B migrated at 65 kD, which corresponds to
the molecular mass of the p65NF-B subunit. Stripping and
reprobing the membrane with a p65NF-B subunit antibody in-
deed confirmed the presence of this subunit at 65 kD (Fig. 4,
compare B [right] with C [right]). To assess specific protein–
protein interactions of BiotinhbdPRELP with our candidate pro-
teins, we performed immunoprecipitation assays in prefusion
osteoclasts treated with vehicle or with BiotinhbdPRELP, dem-
onstrating that indeed the tagged peptide coimmunoprecipitated
in a complex with annexin II (Fig. 4 E) as well as in a complex
with p65NF-B (Fig. 4 F).
Role of annexin II
We next asked what the role could be of annexin II in hbdPRELP
activity. Fig. 5 A (a) shows that BiotinhbdPRELP colocalized with
annexin II at the cell surface and at endosomes. Interestingly,
annexin II also fully colocalized with chondroitin sulfate
(Fig. 5 A, b). Indeed, incubation with an anti–annexin II antibody
reduced intracellular levels of BiotinhbdPRELP in prefusion
osteoclasts vitally treated with the peptide (Fig. 5 B). Consistent
with the lack of binding to CD44 (Fig. 4 C), an anti-CD44 anti-
body failed to affect the ability of prefusion osteoclasts to inter-
nalize BiotinhbdPRELP (Fig. 5 B).
Role of the NF-B transcription factor
The transcription factor NF-B is a crucial determinant of osteo-
clastogenesis. In prefusion osteoclasts, obtained by incubation
of bone marrow macrophages with M-CSF and RANKL for 4 d,
p65NF-B was translocated to the nucleus (Fig. 5 C, top), and,
in contrast to RANKL withdrawal (Fig. 5 C, middle), treatment
with hbdPRELP did not displace p65NF-B from this localiza-
tion (Fig. 5 C, bottom). This suggests that hbdPRELP does not
affect p65NF-B trafficking but may rather affect its transcrip-
tional activity. To test this hypothesis, we evaluated p65NF-B
binding to DNA using a colorimetric assay (TransAM NF-B
p65 kit), which revealed that this binding was reduced by 50%
in hbdPRELP-treated prefusion osteoclasts compared with con-
trol (Fig. 5 D). Accordingly, luciferase activity assay performed
in the immortalized osteoclast precursors cell line RAW264.7,
which also internalized BiotinhbdPRELP (see Fig. 8), evidenced
a significant reduction of specific p65NF-B transcriptional ac-
tivity after treatment with hbdPRELP (Fig. 5 E).
Expression of osteoclast genes and activity
of signal molecules
To confirm that NF-B activity was impaired by the treatment
with hbdPRELP, we evaluated the transcriptional expression of
downstream osteoclast-specific genes. Real time RT-PCR showed
down-regulation of cathepsin K, CTR (calcitonin receptor),
MMP-9 (metalloproteinase-9), rank, and TRAcP (tartrate-
resistant acid phosphatase) mRNAs (Fig. 6 A). Likewise,
hbdPRELP reduced the expression of genes implicated in cell
fusion such as DC-STAMP and CD44, with a modest effect also
on MFR (macrophage fusion receptor; Fig. 6 A).
We next evaluated the effect of hbdPRELP on MAPK signal-
ing. Fig. 6 B shows that this treatment did not prevent RANKL-
induced phosphorylation of the MAPKs ERK1/2, JNK, and p38,
suggesting no effect of hbdPRELP on immediate cell signaling
pathways triggered by RANK activation. hbdPRELP also failed to
affect cell survival. In particular, the nuclei of hbdPRELP-treated
prefusion osteoclasts, stained with DAPI, showed no nuclear
fragmentation or altered morphology, indicating that apoptosis
was not induced by treatment with hbdPRELP (Fig. 6 C). Consis-
tently, Western blot analysis showed no changes in protein ex-
pression of the prosurvival factor Bcl-2 and of the proapoptotic
factor Bax nor activation of procaspase 3 (Fig. 6 D).
Specificity
To assess species cross-reactivity, we evaluated the effect of
hbdPRELP on mouse, rat, and human osteoclastogenesis. As shown
in Fig. S2, prefusion osteoclasts of all three species showed
similar nuclear internalization of Alexa Fluor 488–hbdPRELP
(left) and inhibition of osteoclast formation (right). Fig. 7 (A–D)

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675 PRELP and osteoclastogenesis • Rucci et al.
Figure 4. Internalization of hbdPRELP and interacting proteins. (A) Vital incubation of prefusion (a–c) or mature mouse osteoclasts (OC; d) with Biotin
hbdPRELP for the minutes indicated. Arrows indicate plasma membrane localization (a), localization in endosomal vesicles (b), and localization in vesicles in
the vicinity of the nucleus (c). N, nuclei. (e) Negative control in the absence BiotinhbdPRELP. (f–h) Prefusion osteoclasts were incubated with BiotinhbdPRELP for
20 min at 37°C (f) or at 4°C (g) or were pretreated with 0.45 U/ml chondroitinase ABC (h) before the incubation with BiotinhbdPRELP. (f and g) Insets show
higher magnification of the framed fields. (i) Fixed and permeabilized prefusion osteoclasts were incubated with Alexa Fluor 488–hbdPRELP. The solid lines
indicate the cell surface, and the dashed lines indicate the nuclear envelope. (B) A prefusion osteoclast lysate was subjected to SDS-PAGE and processed
with BiotinhbdPRELP (BhbdPRELP) as described in Materials and methods. Asterisks indicate nonspecific bands. (C) The filter shown in B was stripped and
Western blotted for annexin II, CD44, and p65NF-B. (D–F) Immunoprecipitation (IP) of BiotinhbdPRELP-treated prefusion osteoclast lysates with preimmune
serum (D), an annexin II antibody (E), or a p65NF-B antibody (F). Results are representative of three independent experiments. PIs, preimmune serum;
TCL, total cell lysate; WB, Western blot.

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JCB • VOLUME 187 • NUMBER 5 • 2009 676
Figure 5. Role of annexin II and NF-B. (A, a) Prefusion osteoclast incubation with BiotinhbdPRELP (BhbdPRELP; 10 min) and immunofluorescence detection
of annexin II. (b) Immunofluorescence detection of chondroitin sulfate and annexin II. (B) Vital incubation of prefusion osteoclasts with BiotinhbdPRELP in
the presence of anti-CD44, anti–annexin II antibody, and an irrelevant IgG. Biotin-594 streptavidin (streptav) served as a negative control in the absence
of BiotinhbdPRELP. (C) Prefusion osteoclasts, treated as indicated, were incubated with an anti–p65NF-B antibody followed by incubation with

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677
PRELP and osteoclastogenesis • Rucci et al.
Young et al., 2005; Majumdar et al., 2008). Interestingly, the
heparin-binding domain representing our hbdPRELP has no sites
of cleavage by these enzymes, thus suggesting that the intact
protein could be enzymatically processed and release active
peptides encompassing the hbdPRELP sequence, which can then
act as physiological regulators of bone resorption. Of course,
we cannot rule out that the intact protein can be internalized as
such, a circumstance addressable only in a systematic study as
the simple use of a tagged intact PRELP cannot distinguish be-
tween the internalization of the entire sequence versus that of
cleaved peptides.
The level of specificity for the effect of the hbdPRELP is in-
teresting. A different peptide, representing the heparin-binding
domain of chondroadherin, unexpectedly turned out to be in-
effective on osteoclast development, albeit it bound to heparin
and was eluted at higher salt concentrations (0.8 M NaCl) com-
pared with the PRELP peptide (0.6 M NaCl; unpublished data).
Thus, the chondroadherin peptide was chosen as an appropriate
control for the specificity of the hbdPRELP peptide for a distinct
class of cell surface GAG chains. This control peptide did not
bind to the osteoclast precursor cells and did not affect osteoclasto-
genesis. Therefore, it is likely that the effect of the PRELP pep-
tide is restricted to a limited number of cells with a specific cell
surface ligand GAG. The exact specificity of the PRELP peptide
for a distinct pattern of sulfation along the chondroitin sulfate
chain is not known. In view of previous results showing tightest
binding to optimally sulfated heparin (Bengtsson et al., 2000), it
is likely that the interaction involves oversulfated disaccharides.
Because the variability of the chondroitin sulfate chain is not
known, the definition of binding parameters and the identification
of the specific chondroitin sulfate chain involved are important
tasks for future work.
PRELP is expressed by osteoblasts; however, consistent
with its high specificity, at least the hbdPRELP peptide used in this
study does not appear to have any autocrine effect on these cells.
Similarly, bone marrow macrophages and epithelial cell lines
were insensitive to hbdPRELP treatment. In contrast, the peptide
directly affected osteoclast precursors at a late stage of differenti-
ation. At this stage, a heparan sulfate proteoglycan, perlecan, not
directly associated with the cell surface and with known ability to
bind hbdPRELP was scanty or absent albeit expressed at earlier
stages (unpublished data). In a previous study, heparan sulfate
proteoglycans were found in mature osteoclasts (Nakano et al.,
2004), but the lack of effect of heparinase III digestion in the
osteoclastogenesis assays rules out a role of this GAG in the
hbdPRELP peptide activities on osteoclast development.
However, we believe that our study has identified the
mechanisms whereby hbdPRELP impairs osteoclast generation.
We found that the peptide recognizes chondroitin sulfate chains
of cell surface proteoglycans. In a separate series of experi-
ments, we have used an Akubio RAPid-4 acoustic biosensor to
show that chondroitin 6-sulfate (from human nucleus pulposus)
shows that hbdPRELP does not affect osteoblast activity. Alexa
Fluor 488–hbdPRELP did not vitally bind to calvarial osteo-
blasts, nor did hbdPRELP affect alkaline phosphatase (ALP) ac-
tivity, ability to mineralize, production of bone matrix proteins,
and signaling protein phosphorylation. Moreover, the expres-
sion of genes representing different osteoblast functions was also
unchanged (Table S1). Consistently, in vivo hbdPRELP failed to
affect the osteoblast parameters in the tibial secondary spongi-
osa of ovariectomized mice (Fig. 7, E–G).
To assess hbdPRELP specificity on other cell types, we per-
formed vital incubation of mouse bone marrow macrophages
with Alexa Fluor 488–hbdPRELP, which was able to bind the cell
surface at 5 min. However, this binding was not followed by pep-
tide internalization and nuclear transfer at later times (Fig. 8 A).
In contrast, we observed hbdPRELP internalization in RAW264.7
cells, representing mouse osteoclast precursors (Fig. 8 B). Vital
incubation with tagged hbdPRELP of the immortalized cell lines
HEK293 (human epithelial kidney) and MDA-MB231 (human
breast cancer) also demonstrated no peptide internalization
(Fig. 8 B). In addition, the MDA-MB231 cells treated with
hbdPRELP showed no changes in proliferation, migration, and in-
vasion ability compared with vehicle-treated cells (Fig. S3), and
HEK293 cells subjected to luciferase assay showed no sensitiv-
ity of NF-B transcriptional activity to hbdPRELP treatment (not
depicted). These results suggest that hbdPRELP activity is spe-
cific only for certain cells such as osteoclasts of various species
at particular stages of development.
Discussion
We believe that we have identified a novel mechanism control-
ling bone homeostasis, in which a domain of an extracellular
matrix molecule modulates bone breakdown. The studied basic
heparin- and, as identified in this study, chondroitin sulfate–binding
N-terminal domain of PRELP was found to block osteoclast
formation by a direct mechanism affecting prefusion osteoclasts
through inhibition of p65NF-B transcriptional activity. To the
best of our knowledge, the activity of this matrix peptide appears
novel, involving chondroitin sulfate chains at the cell surface,
and may represent an important new determinant to control bone
remodeling and homeostasis. Notably, the intact PRELP had
similar effect on osteoclast formation as the hbdPRELP peptide,
and the peptide showed antiresorptive activity in vivo. These ob-
servations have two important implications: (1) the protein is
likely to play a physiological role in the control of bone remod-
eling, and (2) the peptide has the potential to represent a new
antiresorptive biological agent valuable for therapy.
Intact PRELP was similarly active in impairing osteo-
clastogenesis as the hbdPRELP. We found that a cluster of en-
zymes, which cut proteoglycans of the cartilage and/or bone
matrices, can potentially cleave the intact PRELP (Fig. S4;
Breckon et al., 1999; Li et al., 2004; Nakamura et al., 2004;
FITC-conjugated secondary antibody (NF-B; left) and with propidium iodide (PI; middle) to detect nuclei. (D) Colorimetric p65NF-B DNA binding assay
of prefusion osteoclast lysates treated with vehicle or 15 µM hbdPRELP. (E) RAW264.7 cells were treated with control peptide or hbdPRELP and subjected to
the luciferase assay as described in Materials and methods. (D and E) Results are the mean ± SEM of three independent experiments (*, P < 0.05).
 

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JCB • VOLUME 187 • NUMBER 5 • 2009 678
indeed binds the BiotinhbdPRELP peptide (unpublished data).
Internalization and nuclear localization of BiotinhbdPRELP has now
been demonstrated in osteoclasts and depends on chondroitin sul-
fate because these processes are inhibited by the removal of these
chains. Chondroitin sulfate proteoglycans have not been intensely
investigated in osteoclasts (Li et al., 2002), and to the best of our
knowledge, this is the first indication that they are involved in
peptide internalization and endosome formation.
hbdPRELP is a highly cationic peptide with eight arginines
distributed between aa 4 and 21, and this number of arginines was
observed to be optimal for peptide internalization (Nakase et al.,
2008). In fact, highly cationic peptides are able to enter into the
cytoplasmic compartment and nucleus of cells from the extra-
cellular environment (Futaki et al., 2002; Kosuge et al., 2008;
El-Sayed et al., 2009), although they have no nuclear localization
sequence. A recent study analyzing live cells demonstrated that
these peptides enter through endocytosis and accumulate in
endocytic vesicles without necessarily routing via the cytoplasm
(Futaki et al., 2007). Arginine-rich peptides, including a basic
peptide segment derived from the HIV-1 Tat protein, are catego-
rized into one of the most frequently used peptide vectors (Futaki
et al., 2007), and their uptake is dependent on heparan sulfate and
chondroitin sulfate proteoglycans (Nakase et al., 2007). This is in
line with our study and corroborates our observations that cell
surface chondroitin sulfate chains are indispensable for the mech-
anism of action of hbdPRELP in osteoclasts.
In contrast, BiotinhbdPRELP internalization was totally inde-
pendent of CD44 activity. This type I transmembrane glyco-
protein is expressed by the osteoclast lineage, is involved in fusion
of macrophages, and binds several matrix components, including
hyaluronan, and its occupancy by matrix components prevents the
formation of polykaria (Sterling et al., 1998). However, CD44
mRNA expression is impaired by hbdPRELP treatment, thus con-
tributing to its inhibitory effect through a transcriptional route.
Importantly, our peptide appears to colocalize with an-
nexin II, forming a complex indispensable for internalization in
endosomes and transfer to the nucleus. Annexin II is a calcium-
dependent phospholipid-binding protein that is involved in
early endosomal organization (Harder et al., 1997). Menaa et al.
(1999) suggested that annexin II stimulates osteoclastogenesis
and bone resorption by activating T cells through a putative re-
ceptor to secreted granulocyte M-CSF. In contrast, in our study,
we found a pivotal role for this protein in the inhibitory effect
of hbdPRELP on osteoclast formation through its indispensible
ability to cause peptide internalization. This discrepancy does
not subtract from the results as in the two studies, the cell types
and the phases of osteoclastogenesis in which annexin II was
involved were different and the physiological control of bone
resorption is known to require a balance between stimulatory
and inhibitory stimuli. Collectively, our results point to chon-
droitin sulfate cell surface proteoglycans and annexin II as
Figure 6. Transcriptional regulation, signaling proteins, and apoptosis.
(A) Real-time RT-PCR analyses of mRNA levels in hbdPRELP-treated puri-
fied prefusion osteoclasts relative to cultures treated with vehicle (set to 1;
dashed line). Data, normalized versus the house-keeping gene Gapdh,
are the mean ± SEM of three independent experiments (*, P < 0.05 vs.
vehicle). (B) Purified bone marrow macrophages, treated for 4 d with
M-CSF, were preincubated for 1 h with vehicle or 15 µM hbdPRELP and then
with RANKL for 15 min. Cells were lysed, and Western blot analysis was
performed for the indicated MAPKs. ERK, extracellular signal-regulated
kinase; p, phospho. (C) Nuclei of prefusion osteoclasts treated with vehicle
(top) or with hbdPRELP (bottom) were stained with DAPI to evaluate apopto-
sis. (D) Western blot analysis of prefusion osteoclasts for the detection of
Bcl-2, Bax, and caspase 3 apoptosis-related proteins. Results are represen-
tative of three independent experiments.
 

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679 PRELP and osteoclastogenesis • Rucci et al.
Figure 7. Effect of hbdPRELP on osteoblasts. (A) Calvarial osteoblasts were fixed and incubated for 1 h with Alexa Fluor 488–hbdPRELP and propidium
iodide (PI). (B) Calvarial osteoblasts were incubated for 4 d with vehicle, 15 µM hbdPRELP, or 15 µM of control peptide. Cells were fixed, and ALP was
detected (top) and quantified (bottom). (C) Details of von Kossa staining of mineralized nodules in calvarial osteoblasts treated with vehicle, 15 µM hbdPRELP,
or 15 µM of control peptide (top) and relative quantification of mineralization in the whole cultures (bottom). (D) Western blot analysis of calvarial osteoblast
lysates for the proteins indicated. (A–D) Data are representative of the mean ± SEM of three independent experiments. ERK, extracellular signal-regulated
kinase; OPN, osteopontin; OSAD, osteoadherin; p, phospho. (E–G) Histomorphometric analysis of ovariectomized (OVX) mice treated with vehicle,
10 mg/kg body weight of hbdPRELP or control peptide, and 1 mg/kg body weight of alendronate, as a reference drug, 5 d/wk for 5 wk. (E) Double
in vivo calcein labeling. (E–G) Data are representative or the mean ± SD of five mice per group. ObS/BS, osteoblast surface/bone surface; MAR, mineral
apposition rate.

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JCB • VOLUME 187 • NUMBER 5 • 2009 680
that treatment of prefusion osteoclasts with hbdPRELP does not
affect RANKL-dependent MAPK phosphorylation, suggesting
that the impairment of immediate cell signaling is not involved
in its mechanism of action. It is also interesting that the suppres-
sion of NF-B to around 50% was sufficient to strongly sup-
press osteoclastic differentiation, likely reflecting the fact that
massive NF-B activity is required for full induction of osteo-
clast formation. Furthermore, the PRELP peptide could exert
additional roles at the cell surface by stimulating interactions
with other cell surface molecules.
Notwithstanding the clear effect of hbdPRELP on osteo-
clast formation, we did not find any direct effect on in vitro
bone resorption, as demonstrated in osteoclasts allowed to dif-
ferentiate normally and then transferred onto bone slices and
treated with hbdPRELP. In this regard, it has to be pointed out
that, although the pivotal role of RANKL-induced NF-B sig-
naling is well established for osteoclast formation, the effect of
this signaling on the mechanism of bone resorption remains to
be fully elucidated (Teitelbaum and Ross, 2003).
In conclusion, the heparin-binding domain of PRELP is a
novel direct negative regulator of osteoclast generation, which
is also effective in vivo. It inhibits NF-B signaling in late-stage
crucial determinants bringing about BiotinhbdPRELP endocytosis
and trafficking.
A key element of our findings seems to be the ability of
hbdPRELP to reach the nuclear compartment and form a complex
with the p65 subunit of the transcription factor NF-B. In bone
marrow macrophages, p65NF-B is translocated to the nucleus
after RANK activation by RANKL. RANK appears in late
osteoclast precursors (Teitelbaum and Ross, 2003), which is con-
sistent with our observation that hbdPRELP is active only in late
stages of osteoclastogenesis. Indeed, in our experimental condi-
tions, BiotinhbdPRELP and p65NF-B physically interact with
a consequent reduction of NF-B activity. This transcription
factor is central to the osteoclastogenic process, and deletion
of NF-B subunits leads to blockage of osteoclast formation
(Franzoso et al., 1997). Therefore, our observation favors the
involvement of NF-B in the mechanism whereby hbdPRELP
blocks osteoclastogenesis. Consistently, no sign of apoptosis
was observed in prefusion osteoclasts treated with hbdPRELP.
This observation is coherent with the results by Franzoso et al.
(1997), showing that p52 and p50NF-B double gene inactiva-
tion in mice does not cause osteoclast death but rather halts the
development of osteoclast precursors. It is interesting to note
Figure 8. Specificity of the hbdPRELP effect.
(A) Confocal microscopy of mouse bone marrow
macrophages (mBMMs) vitally incubated for
5, 10, and 20 min with 15 µM Alexa Fluor
488–hbdPRELP. The bottom panels represent
the nuclear DAPI staining of the cells present
in the corresponding top panels. (B) Con-
focal microscopy of mouse osteoclast-like cells
(RAW264.7), human epithelial kidney cells
(HEK293), human breast cancer cells (MDA-
MB231), and mouse prefusion osteoclasts
(PreOCs) vitally incubated for 20 min with
15 µM BiotinhbdPRELP as described in A. The
bottom middle and right panels represent the
nuclear DAPI staining of the cells present in
the corresponding top panels. Results are repre-
sentative of three independent experiments.

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681 PRELP and osteoclastogenesis • Rucci et al.
prefusion committed osteoclast precursors, with no effect on
osteoblasts and other cell types tested. These observations could
open new avenues for the understanding of the biology of osteo-
clasts and the involvement of matrix components in the regula-
tion of bone development and remodeling and could also have
implications for the treatment of bone diseases.
Materials and methods
Peptides
Intact PRELP was extracted and purified from bovine nasal cartilage
(Bengtsson et al., 2000). A synthetic human peptide (NH2-QPTRRPRPGTGPG-
RRPRPRPRPTPC-COOH) corresponding to the 24 N-terminal aa of PRELP and
representing the active part of the heparin-binding domain (hbdPRELP) was
synthesized by Schafer-N with an additional cysteine residue in its C-terminal
end. One preparation of the peptide was synthesized with a biotin at the
C-terminal cysteine (BiotinhbdPRELP), whereas another preparation was re-
acted with Alexa Fluor 488 C5 maleimide (Alexa Fluor 488–hbdPRELP;
Invitrogen) according to the manufacturer’s instructions. The degree of de-
rivatization was estimated to be in excess of 90% and was verified by using
MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) mass
spectrometry. In pilot experiments, we tested a different heparin-binding pep-
tide from chondroadherin of 14 aa (NH2-CKFPTKRSKKAGRH-COOH), cor-
responding to the C-terminal part of chondroadherin. This peptide turned out
to be ineffective on osteoclast development and was used as a control.
Animals
Procedures involving animals and their care were conducted in conformity
with national and international laws and policies (European Economic
Community Council Directive 86/609, OJ L 358, 1, December 12, 1987;
Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica
Italiana no. 40, February 18, 1992; National Institutes of Health guide for
the Care and Use of Laboratory Animals, National Institutes of Health Pub-
lication no. 85-23, 1985) and were approved by the Institutional Review
Board of the University of L’Aquila.
In vivo study
8-wk-old female C57Bl6/J mice were ovariectomized and, after 3 d, were
treated with vehicle (PBS), control peptide (10 mg/kg body weight), hbdPRELP
(10 mg/kg body weight), or alendronate (1 mg/kg body weight) by i.p.
injection 5 d/wk for 5 wk (number of animals/group = 5). At the end of
the experiment, urine samples were collected for detection of CTX assay by
the Ratlaps ELISA kit (IDS Nordic Bioscience) according to the manufactur-
er’s instructions. Animals were then sacrificed, and the hindlimb long bones
were removed and fixed in 4% paraformaldehyde.
Histomorphometric analysis
Tibiae, embedded in glycol-methacrylate (Technovit 9100 New; Heraeus
Kulzer GmbH), were sectioned longitudinally through the frontal plate, and
sections (2 µm thick) were subjected to TRAcP staining. Osteoclast number/
bone surface (number/square millimeters), osteoclast surface/bone surface
(percentage), osteoblast surface/bone surface (percentage), and structural pa-
rameters, including bone volume/total volume (percentage), trabecular num-
ber (number/millimeter), trabecular thickness (micrometers), and trabecular
separation (micrometers), were measured in a metaphyseal region extending
at least 100 µm away from the distal end of the growth plate and excluding
the endocortical surfaces (secondary spongiosa; Parfitt et al., 1987; Marzia
et al., 2000). Dynamic assessment of the mineral apposition rate was calcu-
lated after double injection of calcein 10 and 3 d before animal sacrifice. Histo-
morphometric measurements were performed with an interactive image
analysis system (IAS 2000; Delta Sistemi) consisting of a color video–equipped
computer linked to the microscope by a video camera.
Osteoclast cultures
Bone marrow was flushed from the long bones of 7-d-old CD1 mice. Cells
were recovered and cultured in DME plus 10% FBS up to 6 d in the presence
of 108 M 1,25(OH)2VitaminD3. To obtain purified bone marrow macro-
phages, bone marrow cells from 7-d-old mice or rats were diluted 1:1 in
Hanks’ balanced salt solution, layered over Histopaque 1077, and centri-
fuged at 400 g for 30 min. Cells recovered were resuspended in DME con-
taining 10% FBS and plated. After 3 h, cultures were extensively washed to
remove nonadherent cells, and then DME supplemented with 10% FBS,
50 ng/ml M-CSF, and 120 ng/ml RANKL was added to adherent cells to
induce osteoclastogenesis. Human osteoclasts were differentiated from the
peripheral blood mononuclear cells. In brief, diluted blood (1:1 in Hanks’
solution) was layered over Ficoll/Histopaque 1077 solution and centrifuged
at 400 g for 30 min. Buffy-coat cells thus isolated were washed twice with
Hanks’ solution, resuspended in DME containing 10% FBS, and plated. After
3 h, cells were rinsed to remove nonadherent cells and cultured in the same
medium in the presence of 50 ng/ml M-CSF and 30 ng/ml RANKL. Unless
otherwise stated, treatment of osteoclast cultures with hbdPRELP or control
peptide started the third day of culture, affecting only adherent cells referred
to as prefusion osteoclasts, which are defined as TRAcP-positive mononuclear
cells. The peptide was replaced at each change of medium.
Enzymatic treatments
To assess the involvement of cell surface proteoglycans carrying heparin
sulfate or chondroitin sulfate chains in the inhibitory effect of PRELP, osteo-
clastogenesis was performed in the presence of 2 U/ml heparinase III or
0.45 U/ml chondroitinase ABC, respectively. The enzymes were replaced
at each change of medium. At the end of the experiment, enzymatic activ-
ity was terminated by extensive washing of the cultures, which were then
fixed in 4% paraformaldehyde.
TRAcP activity
Cells were fixed in 4% paraformaldehyde, and then TRAcP activity was
evaluated histochemically using the Sigma-Aldrich kit #386 according to
the manufacturer’s instruction.
Bone resorption
Osteoclasts were differentiated as described in the Osteoclast cultures sec-
tion onto bone slices or were differentiated in plastic dishes, detached by
trypsin procedure, and cultured onto bone slices for 48 h. Slices were then
fixed in 4% paraformaldehyde, ultrasonicated in 1% sodium hypochlorite
to remove the cells, and stained with 1% toluidine blue. Pit index was com-
puted according to Caselli et al. (1997). In brief, the resorption pits were
divided in three visual categories according to their diameter: small, <10 µm;
medium, 10–30 µm; and large, >30 µm. The numbers of pits per each cat-
egory were scored by multiplying by a different factor according to their
dimensions: for small pits, 0.3; for medium pits, 1; and for large pits, 3.
The sum of the three scores gave the pit index.
Osteoblast cultures
Calvariae were removed from 7-d-old CD1 mice and digested three times
with 1 mg/ml Clostridium histolyticum type IV collagenase and 0.25%
trypsin for 20 min at 37°C. Cells from the second and third digestions were
grown in DME plus 10% FBS and checked for the expression of the osteo-
blast markers ALP, Runx2 (Runt-related transcription factor-2), PTHrP (para-
thyroid hormone/parathyroid hormone–related peptide receptor), type I
collagen, and osteocalcin (Marzia et al., 2000).
ALP activity
Osteoblasts were fixed in 4% paraformaldehyde, and then ALP activity
was evaluated histochemically using the Sigma-Aldrich kit #85 according
to the manufacturer’s instruction.
Mineralization assay
Osteoblast standard medium was supplemented with 10 mM -glycero-
phosphate and 50 µg/ml ascorbate. Osteoblasts were cultured for 3 wk
before characterization of mineralization by von Kossa staining.
Adhesion assay
Osteoclast-enriched bone marrow cultures were trypsinized and treated
in suspension for 30 min with vehicle alone or with 15 µM peptide in
FBS-free DME. Cells were then plated in wells coated with 20% FBS. At
the end of the incubation, attached cells were fixed and subjected to
TRAcP histochemical staining. Multinucleated TRAcP-positive osteoclasts
were then enumerated.
Real-time RT-PCR
Total RNA was extracted using the TRIZOL, and then 1 µg was reverse
transcribed, and the equivalent of 0.1 µg was used for the PCR reactions
using the Brilliant SYBR green QPCR master mix (Agilent Technologies).
Primer sequences and real-time conditions are listed in Table S2.
Immunoprecipitation and Western blotting
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer, and pro-
tein content was measured by the Bradford method. For immunoprecipita-
tion, 5 µg of specific antibodies or preimmune serum was incubated for

Page 14

JCB • VOLUME 187 • NUMBER 5 • 2009 682
Online supplemental material
Fig. S1 shows the effect of hbdPRELP on bone resorption and evaluation
of its internalization. Fig. S2 shows the effect of hbdPRELP on mice, rat,
and human osteoclast formation. Fig. S3 shows the effect of hbdPRELP on
MDA-MB231 cell proliferation, migration, and invasion. Fig. S4 contains
a schematic representation of the intact PRELP protein. Table S1 shows the
effect of hbdPRELP on the transcriptional expression of osteoblast genes.
Table S2 lists the primer sequences and real-time conditions used in
this study. Online supplemental material is available at http://www.jcb
.org/cgi/content/full/jcb.200906014/DC1.
We are indebted with Dr. Rita Di Massimo for her excellent assistance in writ-
ing this manuscript.
This work was supported by the European Commission grant OSTEO-
GENE (contract no. LSHM-CT-2003-502941) to A. Teti and D. Heinegård, by
the Swedish Research Council and King Gustaf V´s 80-year fund to D. Heinegård,
and by grants from the Italian Association for Cancer Research and from the
Swiss Bridge Foundation to A. Teti. N. Rucci is the recipient of the Robert
Schenk Research Prize 2009. M. Capulli is the recipient of a Federazione Itali-
ana della Ricerca sul Cancro fellowship. A. Del Fattore is the recipient of a
European Calcified Tissue Society/Amgen fellowship.
Submitted: 3 June 2009
Accepted: 27 October 2009
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2 h at 4°C with protein G–conjugated agarose beads. After five washes in
RIPA buffer, 1 mg of protein of each sample was added and incubated
overnight at 4°C. Samples were then washed with RIPA buffer, resuspended
in 2× reducing Laemmli sample buffer, and boiled before SDS-PAGE.
For Western blots, proteins resolved on a 10% SDS-PAGE were
trans-blotted to nitrocellulose membranes and probed with the primary
antibody for 1 h at room temperature, washed, and incubated with the
appropriate HRP-conjugated secondary antibody for 1 h at room temper-
ature. In Fig. 4 B, blots were first incubated with BiotinhbdPRELP and then
washed and incubated with streptavidin/HRP. Protein bands were re-
vealed by ECL.
p65NF-B binding activity
p65NF-B binding to DNA was evaluated using the ELISA TransAM NF-B
p65 kit #40096 (Active Motif) according to the manufacturer’s instructions.
In brief, the kit consists of a 96-well plate with an immobilized oligonucleo-
tide containing a p65NF-B consensus binding site. The activated
p65NF-B contained in cell extracts specifically bound to this oligonucleo-
tide, and then the complex was detected by an antibody directed against
the p65NF-B subunit. The addition of an HRP-conjugated secondary anti-
body provided sensitive colorimetric readout.
Luciferase assay
RAW264.7 cells were transfected with 1.35 µg of pNF-B–Luc vector
(Takara Bio Inc.), 1.35 µg of pMT2T-p65 vector, and 0.3 µg of pRL-TK vec-
tor using Lipofectamine 2000 (Invitrogen). After 36 h, cells were harvested,
and lysates were incubated with the Luciferase Assay Substrate by using
the Dual-Luciferase Reporter Assay system (Promega) according to the
manufacturer’s instruction. Firefly luciferase activity was normalized to
Renilla luciferase activity.
Proliferation, migration, and invasion assays
Proliferation of MDA-MB231 cells was evaluated using the CellTiter 96
Aqueous One Solution Cell Proliferation Assay (MTS) from Promega. Mi-
gration assay was performed by the modified Boyden chamber method
(Albini et al., 1987). Polycarbonate filters were coated with 45 µg/cm2
gelatin in the upper compartment of the trans-well chambers, and MDA-
MB231 cells were added and allowed to migrate for 12 h in the presence of
NIH3T3 cell–conditioned media. Filters were then stained with hematoxylin/
eosin. Invasion assay was performed in a similar manner except that the
filters were coated with 35 µg/cm2 of reconstituted matrigel and processed
after 24 h.
Fluorescence and confocal microscopy
Cells were fixed with 4% paraformaldehyde and incubated for 1 h at room
temperature with Alexa Fluor 488–conjugated hbdPRELP (Alexa Fluor 488–
hbdPRELP) or primary antibodies followed by FITC- or TRITC-conjugated sec-
ondary antibody. Cells were also incubated vitally with BiotinhbdPRELP,
which was revealed by fluorescent biotin 594-streptavidin. To detect
nuclei, cells were stained with DAPI (blue fluorescence) or with propidium
iodide (red fluorescence). Cells were then observed at room temperature
by conventional epifluorescence using a microscope (Axioplan; Carl Zeiss,
Inc.) or by confocal microscopy using a confocal microscope (FluoView
IX81 FVBF; Olympus). For fluorescence microscopy, we used 2.5× NA
0.075, 10× NA 0.30, 20× NA 0.5, and 40× NA 0.75 Plan-Neofluar ob-
jective lenses. Images were captured with a camera (AxioCam MRC5;
Carl Zeiss, Inc.) using the AxioVs 40 version 4.7.1.0 software (Carl Zeiss,
Inc.). For confocal microscopy, we used 10× NA 0.30 and 40× NA 0.85
UPlan-Apochromat or 60× NA 1.4 oil Plan-Apochromat objective lenses.
Images were captured using FluoView 500 software (Olympus).
Digital images
Light, fluorescence, and confocal microscopy pictures were captured as
JPEG files as specified in the previous section. Western blots images were
captured by the Molecular Analyst software for the model 670 scanning
densitometer (Bio-Rad Laboratories) as JPEG files. The areas of interest
were selected and reproduced for documentation by Photoshop version 6
or 7 software (Adobe).
Statistics
All experiments were performed in triplicates and repeated at least three
times. Data are expressed as the mean ± SEM. Statistical analysis was
performed by one-way analysis of variance, followed by unpaired Stu-
dent’s t test. A p-value of <0.05 was conventionally considered statisti-
cally significant.

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683PRELP and osteoclastogenesis • Rucci et al.
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