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Departments of Endocrinology and Metabolic Diseases (M.M.L.D., M.K., C.v.d.B., S.E.P., C.W.G.M.L.) and Pediatrics (M.K.), Leiden University Medical Center, 2333ZA Leiden, The Netherlands; and Pharmaceutical Research Laboratory, Kirin Brewery Co. Ltd. (T.Y.), 3 Miyahara, Takasaki, Gunma 370-12, Japan
Address all correspondence and requests for reprints to: Dr. Martine M. L. Deckers, Department of Endocrinology and Metabolic Diseases, Albinusdreef 2, Building 1 C4R-89, 2333 ZA Leiden, The Netherlands. E-mail: m.m.l.deckers{at}lumc.nl
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Abstract |
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In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGF165R/neuropilin, coincided with expression of their ligands, being maximally expressed during mineralization. VEGF-A production during osteoblast differentiation was stimulated by insulin-like growth factor I that enhances osteoblast differentiation and was inhibited by PTH-related peptide that inhibits osteoblast differentiation. Furthermore, continuous treatment of KS483 cells with recombinant human VEGF-A stimulated nodule formation.
Although treatment of KS483 cells with soluble FLT1, an agent that blocks binding of VEGF-A and -B to VEGFR1, did not inhibit nodule formation, this observation does not exclude involvement of VEGFR2 in the regulation of osteoblast differentiation. As it is known that VEGF-A, -C, and -D can act through activation of VEGFR2, other isoforms might compensate for VEGF-A loss. The expression pattern of VEGFs and their receptors shown here suggests that VEGFs play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF is a glycoprotein that shares homology to platelet-derived growth factor (12). It acts as both a permeability and an angiogenic factor (13, 14, 15). The VEGF gene can be alternatively spliced, resulting in three distinct gene products corresponding to 120, 164, or 188 amino acids in the mouse, or five distinct gene products, respectively corresponding to 121, 145, 165, 189, and 206 amino acids in humans. Each splice variant has specific characteristics (16, 17). In fetal tissues VEGF messenger RNA (mRNA) expression has been described in various organs, including the skeleton (18, 19).
Recently, other angiogenic factors have been identified that have sequence homology to VEGF, which is now termed VEGF-A. The biological effects of these VEGF family members, named VEGF-B, -C, and -D or c-fos-induced growth factor (FIGF) and placenta growth factor-1 (PlGF-1), -2, and -3 are still not completely elucidated. All members of the VEGF family have in common the ability to stimulate endothelial cell proliferation (20). Similar to VEGF-A, VEGF-B can be alternatively spliced into two different splice variants (VEGF-B167 and VEGF-B186). VEGF-B is thought to play a role in vascularization of skeletal muscle (21), whereas VEGF-C is most likely involved in lymphangiogenesis (20). VEGF-B and VEGF-C are, like VEGF-A, associated with tumor angiogenesis (22). Recently, expression of VEGF-B and VEGF-D mRNA has been shown during tooth and bone development (23, 24). Expression of PlGF is restricted to the placenta (25).
Homo- or heterodimers of the VEGF family act as paracrine factors through signaling via various type III tyrosine kinase receptors (26): 1) VEGF receptor 1 or fms-like tyrosine kinase receptor 1 (FLT-1/VEGFR1), which binds VEGF-A, VEGF-B, and PlGF; 2) VEGFR2 or fetal liver kinase 1 (FLK-1), which binds VEGF-A, -C, and -D with high affinity, but not PlGF (27, 28); 3) VEGFR3 or FLT4, which binds VEGF-C and -D (29, 30). Recently, neuropilin has been identified as a new isoform-specific VEGF receptor (VEGF165R). Neuropilin acts as a coreceptor of VEGFR2 and binds PlGF-2 and the isoform VEGFA165, but not VEGFA121 (31, 32, 33). Whether neuropilin is expressed in osteoblasts is at present unknown.
VEGF was reported to induce alkaline phosphatase activity in primary osteoblasts and to enhance their responsiveness to PTH. Furthermore, high affinity sites for VEGF-A have been identified on primary osteoblasts, indicating the presence of functional VEGFRs on osteoblasts (34). This suggests that besides its established effects on endothelial cells, VEGF might play a role in osteoblast differentiation. In addition, osteotropic factors, such as vitamin D3, insulin-like growth factor I (IGF-I), and PGs, have been shown to increase VEGF-A mRNA expression and VEGF secretion by osteoblast-like cells (35, 36, 37, 38). In this study we examined expression of VEGFs and their receptors in preosteoblast-like cells as well as the potential role of VEGFs during differentiation of KS483 cells, a nontransformed preosteoblast-like cell line.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MEM (Life Technologies, Inc., Breda, The Netherlands) supplemented with
10% FCS (Integro B.V., Zaandam, The Netherlands) and
penicillin/streptomycin (Life Technologies, Inc.). Three
days after plating the cells reached confluence and were subsequently
cultured in presence of 50 µg/ml ascorbic acid and in the presence or
absence of growth factors [PTH-related peptide (PTHrP)-(1–34);
Bachem, Heidelberg, Germany], recombinant human (rh)
VEGF165, rhIGF-I (Santa Cruz Biotechnology, Inc., Breda, The Netherlands) or goat antimouse VEGF
neutralizing antibody (R&D Systems, Uithoorn, The Netherlands).
Truncated VEGFR1 or soluble fms-like tyrosine kinase receptor 1
(sFLT1) was provided by Dr. H. Weich (National Center for
Biotechnology, Braunschweig, Germany). The medium was replaced every
3–4 days. When nodules appeared (between 14–18 days after plating),
medium was replaced by medium containing 5 mM
ß-glycerophosphate and ascorbic acid to further stimulate osteogenic
differentiation. At different time points, supernatants and cell layers
were collected separately. VEGF-A production and alkaline phosphatase
activity were determined in the culture supernatants, whereas mRNA,
DNA, and protein expression were determined in the cell layer.
Nodule formation
After 18 days of culture the number of nodules was counted in
control cultures and in VEGF-A-, IGF-I-, PTHrP-, or sFLT1-treated
cultures by light microscopy at a 40-fold magnification. Cultures were
performed in quadruplicate, and three fields per well were
counted.
DNA assay
At the end of each culture period the supernatants were
aspirated, and the cells were washed twice with PBS. DNA was released
from the cells by freeze-thawing in 1 x SSC (standard saline
citrate) buffer containing 0.01% SDS. DNA content was determined using
Hoechst stain 2495 (ICN Biomedicals, Inc., Zoetermeer, The
Netherlands) and calibrated against a DNA standard of 0.5–10 µg/ml
DNA. Samples were excited with light (460 nm), after which fluorescent
emission was measured at 365 nm.
VEGF-A enzyme-linked immunosorbent assay (ELISA)
VEGF-A levels in culture supernatants were assayed with a
quantitative sandwich ELISA (R&D Systems) according to the
manufacturer’s instructions. Values were assayed in triplicate and
corrected for DNA content. The sensitivity of the assay is 3 pg/ml, and
it detects both VEGF120 and
VEGF164. Cross-reactivity with human
VEGF165, murine platelet-derived growth factor,
or murine PlGF was less than 0.05%. Cross-reactivity with other
members of the VEGF family is not known.
RNA isolation
The cells were lysed in a solution of 4 M
guandinium thiocyanate containing 25 mM sodium citrate (pH
7.0), 0.5% sarkosyl, and 0.1 M 2-mercaptoethanol, and
total RNA was extracted by the addition of phenol and chloroform
followed by precipitation with isopropanol as previously described
(41). The concentration of the isolated RNA was determined by UV
absorption spectrophotometry at a wavelength of 260 nm. RNA quality was
checked on an agarose gel containing 0.5 µg ethidium bromide per
ml.
Competitive RT-PCR
Total RNA (1 µg) was reversed transcribed into
complementary DNA (cDNA) as previously described (42). A RT-PCR mixture
was prepared that contained first strand buffer (50 mM
Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM
MgCl2), 5 mM dithiothreitol, 200 ng
random hexanucleotides, 2.5 U murine Moloney leukemia virus reverse
transcriptase, 0.375 mM deoxy-NTPs (Life Technologies, Inc.), and 1 U RNAsin (Promega Corp.,
Leiden, The Netherlands). To correct for differences in cDNA levels,
4-fold serial dilutions of an internal standard plasmid pMUS that
encode for the housekeeping gene
ß2-microglobulin
(ß2-MG) were coamplified with 5 ng sample cDNA
by competitive RT-PCR. The synthetic internal standard pMUS was
provided by Dr. D. Shire (Sanofi Pharmaceuticals, Inc.,
Labege, France). This method has been described in detail by Van
Bezooijen et al. (42). The PCR reaction was carried out in a
Hybaid Omnigene thermal cycler (Biozym, Landgraaf, The Netherlands).
cDNA was denatured at 94 C for 3 min, followed by repeated cycles
consisting of 94 C for 30 sec, 56 C for 30 sec, and 72 C for 1 min. For
quantification of ß2-MG levels, cDNA was
amplified over 33 cycles. Gels were photographed under UV light and
analyzed by computerized densitometry. Samples were regarded to contain
equal amounts of cDNA at a ratio of cDNA to construct of 1, as
described previously (42) (Fig. 1
).
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Primers for VEGFR1 and VEGFR2 mRNA were used as reported by Fong
et al. (43), whereas other primers were designed using
Primer III Design software (http//www.genome.wi. mit.edu) and
checked against GenBank to avoid cross-reactivity with other known
sequences. A PCR mixture was prepared using 20 ng cDNA/reaction, PCR
buffer [10 mM Tris-HCl (pH 9.6), 50
mM NaCl, and 0.2 mg BSA/ml; Eurogentec, Seraing,
Belgium], 0.25 µM oligonucleotide primers
(Eurogentec), 1.5 mM MgCl2,
0.2 mM deoxy-NTP (Promega Corp.),
and 0.5 U Goldstar DNA polymerase/µl (Eurogentec). The PCR reactions
were carried out in a Hybaid Omnigene Thermal cycler (Biozym,
Landgraaf, The Netherlands). cDNA was denatured at 94 C for 3 min,
followed by repeated cycles to amplify the cDNA consisting of 94 C for
30 sec, 58 or 60 C for 30 sec, and 72 C for 1 min. PTH/PTHrP receptor
and VEGF-B were amplified over 30 cycles. BSP, osteocalcin, VEGF-A,
VEGF-B, VEGF-D, FLT1, and VEGFR2/FLK1 cDNA were amplified over 35
cycles, VEGFR165/neuropilin over 30 and 35 cycles, and VEGF-C and FLT4
over 38 cycles. Samples and a 100-bp ladder were electrophoresed on a
1.5% agarose gel containing 0.5 µg ethidium bromide/ml. PCR
reactions were repeated at least three times. Relative mRNA levels of
three separate experiments were determined by densitometry. Bands with
the highest intensity were regarded as 100%.
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Statistics
Results are depicted as the mean ± SEM.
Differences between groups were determined by one-way ANOVA for
multiple comparison, followed by Fisher’s least significant difference
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. As shown in Fig. 2A, PTH/PTHrP was expressed at low levels
7 days after plating, and this expression increased during further
differentiation. The differentiation markers BSP and OC were expressed
at low levels when nodules appeared (14 days after plating) and
increased during further differentiation (Fig. 2, B and C). In the same
experiment mRNA expressions of VEGF-A isoforms were compared, with the
expression of osteoblast differentiation markers. For this, primers
were designed that can hybridize to three different splice variants of
the mouse VEGF-A gene (VEGF-A120,
VEGF-A164, and VEGF-A188),
resulting in the amplification of VEGF-A120 (230
bp) and VEGF-A164 (360 bp) mRNA levels. In
parallel with PTH/PTHrP receptor mRNA expression,
VEGF-A120 and VEGF-A164
mRNA were expressed at low levels early in differentiation (7 days
after plating) and were strongly increased during mineralization, when
BSP and OC were abundantly expressed (Fig. 3A). The matrix-bound isoform
VEGF-A188, was not expressed by KS483 cells, but
was expressed by fetal liver, which was used as a positive control
(data not shown).
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). In line with VEGF-A mRNA expression, VEGF-B mRNA expression
was expressed at low levels 7 days after plating; then its expression
increased, and it was maximally expressed 21 days after plating (Fig. 3B). VEGF-D mRNA expression paralleled VEGF-B mRNA expression; it was
expressed at low levels early in differentiation and highly expressed
during mineralization (Fig. 3D). In contrast, expression of VEGF-C was
only detected at very low levels after 21 days of culture. This was not
due to suboptimal PCR conditions, because in fetal lung, which was used
as a positive control, VEGF-C was abundantly expressed (Fig. 3C).
Expression of VEGF receptors during differentiation of KS483
cells
VEGFR1, VEGFR2, and VEGF165R/neuropilin, but not VEGFR3, were
expressed in a differentiation-dependent manner (Fig. 3, E–H). Low
levels of VEGFR1 were only detected in differentiating osteoblasts
during the mineralization phase (18 and 21 days after plating). VEGFR2
mRNA expression was low from days 7–14 and was strongly increased at
the start of mineralization (Fig. 3F).
VEGF165R/neuropilin mRNA was maximally expressed
during the mineralization phase (Fig. 3G). The gradual increase in
VEGF165R/neuropilin mRNA expression was confirmed
using 30 cycles of amplification (data not shown). VEGFR3 mRNA
expression was not detected in these cultures even after 38 cycles of
amplification, although in fetal lung, which was used as a positive
control, VEGFR3 mRNA expression was found (Fig. 3H).
VEGF-A production and VEGFR2 protein expression during
differentiation of KS cells
Concurrently with the rise in mRNA expression, VEGF-A protein,
measured by ELISA, accumulated in the culture medium (Fig. 4A). The levels significantly rose from
245 pg/ml in confluent cultures (3 days after plating) to 750 pg/ml
during the mineralization phase (21 days after plating; Fig. 4A). It is
important to note that the unconditioned culture medium (MEM
containing 10% FCS) did not contain detectable levels of VEGF-A
protein (<3.0 pg/ml). In contrast to VEGF-A, there are no ELISAs
commercially available to measure VEGF-B and D levels.
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). In line with the PCR data, VEGFR2 expression
was low at the beginning of the culture and increased, with
differentiation being maximally expressed during the mineralization
phase. Under reducing conditions VEGFR2 migrated as two bands of
approximately 220 and 230 kDa (Fig. 4B). Using fetal lung as a positive
control, proteins of identical size were detected, which most likely
represent differentially glycosylated VEGFR2s. In contrast to
VEGFR2 protein levels, we did not analyze VEGFR1 and VEGFR3
protein expression, because PCR data showed that VEGFR1 mRNA
and 3 mRNA levels are very low or below the detection limit of PCR,
whereas VEGFR2 increased with differentiation.
Effects of IGF-I and PTHrP on VEGF-A production by KS483 cells
Continuous treatment of KS cells with IGF-I stimulated
osteoblast differentiation dose dependently, as shown by a significant
increase in nodule formation after 21 days of culture (Fig. 5A). In contrast, continuous treatment
with PTHrP significantly inhibited nodule formation after 21 days of
culture (Fig. 5A). VEGF levels were determined in the culture medium
after 21 days of culture. IGF-I treatment significantly increased
VEGF-A levels, whereas PTHrP treatment significantly decreased VEGF-A
levels (Fig. 5B).
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, A and B, treatment with 10 and 25
ng/ml rhVEGF-A stimulated nodule formation to a similar extent as
IGF-I. Treatment of cultures with a functional blocking antibody
directed against murine VEGF-A in the presence or absence of IGF-I did
not have an effect on alkaline phosphatase activity (data not shown).
Furthermore, treatment of KS483 cells with 50, 100, and 200 ng/ml
truncated VEGFR1 (sFLT1) did not affect nodule formation 21 days after
plating (Fig. 5C). It should be noted that sFLT1 is able to block
endothelial proliferation and endothelial sprouting in vitro
at the concentrations used (our unpublished results). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteoblast differentiation in vitro is characterized by temporal and sequential gene expression, resulting finally in calcium deposition (39, 49). We show here that VEGF-A, -B, and -D are expressed at low levels at the beginning of osteoblast differentiation before BSP and OC mRNA expression. VEGF-A, -B, and -D mRNA expression strongly increased during terminal differentiation in parallel with BSP and OC mRNA expression and were maximally expressed during mineralization. In line with the mRNA expression of VEGF-A, maximum protein levels were found by ELISA during the mineralization phase. When KS483 cells were treated with the osteotropic factors IGF-I and PTHrP, a correlation between osteoblast differentiation and VEGF production was found. Continuous treatment of KS483 cells with PTHrP, a condition that is known to inhibit osteoblast differentiation (50, 51), also inhibited VEGF-A production significantly. In contrast, IGF-I, which stimulated differentiation of KS483 cells, as shown by enhanced nodule formation, significantly and dose dependently increased VEGF pro- duction.
It is well known that members of the VEGF family act as paracrine factors through activation of VEGFRs present on endothelial cells. Early observations by Midy and Ploet (34) showed binding of labeled VEGF to high affinity sites on bovine calvarial osteoblasts. Recently, VEGFR1 mRNA expression was reported in murine trabecular osteoblasts (11). In agreement with these studies we demonstrated VEGFR1 mRNA expression by murine KS483 cells. Moreover, this expression was dependent on the differentiation state of the cells. VEGFR2 mRNA expression has been shown in intersegmental regions of vertebrae by in situ hybridization (19, 52, 53). We found that VEGFR2 mRNA and protein were expressed in a differentiation-dependent manner. VEGFR2 protein was detected, by immunoblotting, as a 230-kDa mature protein that corresponds to the molecular mass of the mature form of VEGFR2 that is involved in receptor signaling (54). Other studies, however, failed to demonstrate VEGFR2 mRNA in postnatal human trabecular osteoblasts (36) or showed very low VEGFR2 mRNA expression in postnatal mouse trabecular osteoblasts in vivo (11). Possible explanations for the differences between our and the other observations include 1) age difference (fetal vs. postnatal), as VEGFR2 mRNA levels are described to decline postnatally (53); 2) difference of origin (calvarial bone vs. trabecular bone); and 3) cell line-specific properties (cell line vs. primary cells). The most recently identified VEGF receptor, VEGF165R/neuropilin, was also expressed in a differentiation-dependent manner. To our knowledge we are the first to report expression of this receptor in osteoblast-like cells. This receptor has been identified on endothelial cells and tumor-derived cell lines and acts as an isoform specific coreceptor of VEGFR2 by specifically binding the isoform VEGF-A165, but not VEGF-A121 (31, 32). These findings combined with the observation of limb deformities in mice overexpressing neuropilin support a role for this receptor in bone physiology (55).
Treatment of KS483 cells with rhVEGF-A stimulated nodule formation. Interestingly, the effects of rhVEGF-A on differentiation were noted during mineralization at the time when VEGFRs are maximally expressed. These findings, therefore, suggest that VEGF-A might stimulate osteoblast differentiation in an autocrine manner. However, treatment of KS cells with a neutralizing antibody directed against murine VEGF-A or with a truncated VEGF receptor (sFLT1) that can bind both VEGF-A and –B, thereby neutralizing their action, did not affect nodule formation in unstimulated cultures, although both agents were able to inhibit angiogenesis in an in vitro model using fetal mouse bone explants (our unpublished results). This suggests that enhanced VEGF-A production by terminal differentiated osteoblasts is not an essential step in the regulation of nodule formation in vitro, but that it is instead a marker of osteoblast differentiation. However, we cannot exclude involvement of other VEGF family members in osteoblast differentiation, because VEGF-A loss might be compensated by both VEGF-B and VEGF-D, which are also expressed in a differentiation-dependent manner, as shown here. Observations made in sFLT1-treated cultures make it less likely that VEGF-B can compensate for VEGF-A loss, because both VEGF-A and VEGF-B act through the same receptor (VEGFR1). As it is known that VEGF-A can act not only through VEGFR1 but also through VEGR2, VEGF-A loss might be compensated by VEGF-C or VEGF-D, as these isoforms act through binding of VEGFR2.
The expression pattern of VEGFs and their receptors by KS483 cells before mineralization shown here combined with the recent observation that VEGF substitution can rescue osteoclast formation in mice deficient in osteoclasts due to the absence of macrophage colony-stimulating factor (56) support a novel role for VEGFs in the regulation of bone remodeling. Osteoblast-derived VEGFs may not only act as chemotactic factors on endothelial cells, but may also act on osteoclasts to stimulate their formation and activity.
In conclusion, we show here for the first time that VEGF-A, -B, and -D mRNA and, to a lesser extent, VEGF-C are expressed by the murine osteoblast-like KS483 cell line in a differentiation-dependent manner. Furthermore, exogenously added VEGF-A enhances osteoblast differentiation. VEGF-A may act through various VEGFRs (VEGFR1, VEGFR2, and neuropilin) that are also expressed by osteoblasts in a differentiation-dependent fashion. Continuous treatment of KS483 cells with IGF-I and PTHrP, which modulate osteoblast differentiation, is associated with a change in VEGF-A production. We therefore propose that osteoblast-derived VEGFs may act as paracrine factors, modulating endothelial and osteoclast functions, but may also act as autocrine factors, modulating osteoblast differentiation. These observations might be of importance in processes such as endochondral bone formation and fracture healing, where a close association between angiogenesis and bone formation exists.
Received July 5, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. Gurlek, M. R. Pittelkow, and R. Kumar Modulation of Growth Factor/Cytokine Synthesis and Signaling by 1{alpha},25-Dihydroxyvitamin D3: Implications in Cell Growth and Differentiation Endocr. Rev., December 1, 2002; 23(6): 763 - 786. [Abstract] [Full Text] [PDF]
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 J. Street, M. Bao, L. deGuzman, S. Bunting, F. V. Peale Jr., N. Ferrara, H. Steinmetz, J. Hoeffel, J. L. Cleland, A. Daugherty, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover PNAS, July 23, 2002; 99(15): 9656 - 9661. [Abstract] [Full Text] [PDF]
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M. M. L. Deckers, R. L. van Bezooijen, G. van der Horst, J. Hoogendam, C. van der Bent, S. E. Papapoulos, and C. W. G. M. Lowik Bone Morphogenetic Proteins Stimulate Angiogenesis through Osteoblast-Derived Vascular Endothelial Growth Factor A Endocrinology, April 1, 2002; 143(4): 1545 - 1553. [Abstract] [Full Text] [PDF]
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N. Akeno, J. Robins, M. Zhang, M. F. Czyzyk-Krzeska, and T. L. Clemens Induction of Vascular Endothelial Growth Factor by IGF-I in Osteoblast-Like Cells Is Mediated by the PI3K Signaling Pathway through the Hypoxia-Inducible Factor-2{alpha} Endocrinology, February 1, 2002; 143(2): 420 - 425. [Abstract] [Full Text] [PDF]
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 G. R. Beck Jr., B. Zerler, and E. Moran Gene Array Analysis of Osteoblast Differentiation Cell Growth Differ., February 1, 2001; 12(2): 61 - 83. [Abstract] [Full Text]
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K. Pavelock, K. M. Braas, L'H. Ouafik, G. Osol, and V. May Differential Expression and Regulation of the Vascular Endothelial Growth Factor Receptors Neuropilin-1 and Neuropilin-2 in Rat Uterus Endocrinology, February 1, 2001; 142(2): 613 - 622. [Abstract] [Full Text] [PDF]
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