Calcif Tissue Int (2006) 79:360 369 DOI: 10.1007/s00223-006-0099-y
Transient Exposure to PTHrP (107-139) Exerts Anabolic Effects through Vascular Endothelial Growth Factor Receptor 2 in Human Osteoblastic Cells In Vitro A. R. de Gorta´zar,1 V. Alonso,1 M. V. Alvarez-Arroyo,2 P. Esbrit1 1 2
Laboratorio de Metabolismo Mineral y O´seo, Fundacio´n Jime´nez Dı´ az (Capio Group), Avda. Reyes Cato´licos 2, 28040 Madrid, Spain Laboratorio de Metabolismo Renovascular, Fundacio´n Jime´nez Dı´ az (Capio Group), Avda. Reyes Cato´licos 2, 28040 Madrid, Spain
Received: 3 April 2006 / Accepted: 4 August 2006 / Online publication: 14 November 2006
Abstract. Intermittent administration of the N-terminal fragment of parathyroid hormone (PTH) and PTH-related protein (PTHrP) induces bone anabolic effects. However, the effects of the C-terminal domain of PTHrP on bone turnover remain controversial. We examined the putative mechanisms whereby this PTHrP domain can affect osteoblastic differentiation, using human osteosarcoma MG-63 cells and osteoblastic cells from human trabecular bone. Intermittent exposure to PTHrP (107-139), within 10-100 nM, for only £24 hours during cell growth stimulated alkaline phosphatase (ALP) and Runt homology domain protein (Runx2) activities as well as osteocalcin (OC) and osteoprotegerin (OPG) expression but inhibited receptor activator of nuclear factor jB (NF-jB) ligand. Continuous exposure to this PTHrP peptide reversed these effects. The stimulatory effects of transient treatment with PTHrP (107-139) on OC mRNA and/or OPG protein expression were unaffected by a neutralizing anti-insulin-like growth factor I antibody or [Asn10, Leu11, DTrp12]PTHrP (7-34) in these cells. On the other hand, the former antibody and the latter PTHrP antagonist abrogated the PTHrP (1-36)-induced increase in these osteoblastic products. Transient exposure to PTHrP (107-139), in contrast to PTHrP (1-36), stimulated vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in these cells. Moreover, induction of ALP activity as well as OC and OPG expression by PTHrP (107-139) was blunted by SU5614, a permeable tyrosine kinase inhibitor of VEGFR2. Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) inhibitors abolished the PTHrP (107-139)-stimulated VEGFR2 and OPG mRNA levels in these cells. These results indicate that intermittent exposure to PTHrP (107-139) exerts potential anabolic effects through the PKC/ERK
The first two authors contributed equally to this work.This work was presented in part at the International Conference on Progress in Bone and Mineral Research, November 27 29, 2003, Vienna, Austria (published in Bone 33:S17, 2003); at the XLI Congress of the European Renal Association, May 15 18, 2004, Lisbon, Portugal; and at the 26th Annual Meeting of the American Society for Bone and Mineral Research, October 1 5, 2004, Seattle, WA (published in J Bone Miner Res 19[suppl 1]:S194, 2004). Correspondence to: P. Esbrit; E-mail:
[email protected]
pathway and, subsequently, VEGFR2 upregulation in vitro in human osteoblastic cells. Key words: Human osteoblastic cell — Osteoblast differentiation — C-terminal parathyroid hormone-related protein — Vascular endothelial growth factor receptor 2
Parathyroid hormone-related protein (PTHrP), which is produced by a broad spectrum of normal tissues including bone, is now emerging as an autocrine/paracrine regulator of cell growth and differentiation in many of these tissues [1]. Present evidence supports the hypothesis that PTHrP is an important regulator of bone cell function. PTHrP is expressed in both chondrocytes and osteoblasts during bone development, consistent with its important role in endochondral bone formation [1]. In fact, homozygous mice with targeted disruption of the gene for PTHrP or that of its PTH1 receptor (PTH1R) show a lethal chondrodysplasia in the perinatal period [2, 3]. High levels of both PTHrP and PTH1R are present in actively differentiating osteoblasts, suggesting a modulatory role of the PTHrP/PTH1R system in osteoblastic differentiation [4 7]. However, conflicting results have been obtained when assessing the effects of PTHrP on osteoblastic growth and differentiation in different osteoblast-like cells in vitro [8 12]. These discrepancies might be, at least in part, due to differences in either the osteoblastic cell types or the mode of exposure to PTHrP. In this regard, it is now clear that continuous exposure to the N-terminal fragment of PTH or PTHrP in vivo, as occurs in patients with hyperparathyroidism or humoral hypercalcemia of malignancy, induces bone catabolic effects [13]. Meanwhile, its intermittent administration increases bone formation in both humans and rats with osteoporosis [14 16]. The mechanisms of the bone anabolic action of PTH and PTHrP in vivo appear to be complex and might involve their
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interaction with local osteogenic factors such as insulinlike growth factor (IGF)-I [17, 18]. In addition, various in vitro and in vivo studies in rodents indicate that the Cterminal PTHrP domain containing the 107 111 epitope, referred to as osteostatin, is a potent inhibitor of bone resorption, but its true effect in bone in vivo is currently unclear [15, 19, 20]. In this regard, a recent preliminary report has shown that knockin mice with deletion of the C-terminal region of PTHrP have premature osteoporosis associated with decreased osteoblastic bone formation [21]. PTHrP (107-139) can directly interact with osteoblasts, inducing either inhibitory or stimulatory effects on osteoblastic proliferation and/or differentiation in various osteoblastic cell preparations and experimental settings [10 12, 22, 23]. Using both transformed and nontransformed human osteoblastic cells in vitro, we previously demonstrated that this PTHrP domain, in a similar manner to that of the N-terminal PTHrP domain, rapidly induces expression of vascular endothelial growth factor (VEGF), a potent angiogenic and emerging osteogenic factor [24, 25]. These effects of this putative PTHrP fragment appear to occur through a specific receptor different from PTH1R [11, 23]. Several cell model systems have previously been established to characterize the mechanisms underlying the effects of the N-terminal fragment common to PTH and PTHrP on bone formation [26 28]. By using a similar approach to that used in one of these in vitro models, we presently assessed the influence of exposure time on the effects of PTHrP (107 139) on osteoblast differentiation in human osteosarcoma MG63 cells, a useful model for their nontransformed counterparts [8, 25, 29, 30]. Some of the effects of this peptide in MG-63 cells were confirmed in primary cultures of osteoblastic cells from human trabecular bone (hoB cells). These hOB cell types have previously been shown to respond to both N- and C-terminal PTHrP peptides [8, 25, 30]. We explored the putative mechanism involved in the actions of PTHrP (107139) in this in vitro cell system.
Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-p42/p44 extracellular signal-regulated kinases (ERK 1/2) and anti-phospho(p)ERK 1/2 (Thr202/Tyr204) antibodies were supplied by Cell Signaling Technology (Beverly, MA). T4 polymerase was supplied by Promega (Madison, WI). [c32P]Adenosine triphosphate (ATP, 3,000 Ci/mmol) was from Amersham (Aylesbury, UK). Bisindolylmaleimide I (BIM) and 2¢-amino-3¢-methoxyflavone (PD098059), calphostin C, and SU5614 were obtained from Calbiochem (San Diego, CA). Cell Cultures and Treatments MG-63 cells (ATCC CRL 1427) were grown in DulbeccoÕs modified EagleÕs medium (DMEM) with 10% fetal bovine serum (FBS), 1% nonessential amino acids, and antibiotics (100 IU/mL of penicillin, 100 lg/mL of streptomycin), in 5% CO2 at 37C, as described [25, 30]. hOB cells were isolated from trabecular bone explants obtained from knee or hip samples discarded at the time of surgery on osteoarthritic subjects (aged 61 84 years), as previously reported [7, 10, 25, 30, 31]. Cells were cultured in DMEM with 15% FBS and antibiotics and used at passages 1 4. Cells were seeded at 20,000/cm2 in culture medium in six-well plates and refed with fresh medium after 24 hours (day 1). In the intermittent exposure protocol, cells were incubated with each PTHrP peptide for the first 6 or 24 hours of each consecutive 48 hour incubation cycle (up to three cycles). After these treatment periods, fresh medium without PTHrP peptides was added for the remainder of the cycle. In the continuous exposure protocol, cells were exposed to PTHrP peptides from day 1 to the end of the culture period. During this period, culture medium was replaced every 48 hours (in both types of exposure protocols). Control cells were submitted to matching changes of culture medium containing vehicle for each type of protocol used. PTHrP (1-36) and PTHrP (107-139) were dissolved at 0.1 mM in 10 mM acetic acid and 50 mM KCl, adjusted to pH 4.5 with acetic acid, respectively; and further dilutions in culture medium were made for the experiments. In some experiments, an anti-IGF-I antibody (at a final 1:100 dilution), PTHrP (734) (at 1 lM), or the different inhibitors were added 1 hour before addition of PTHrP (107-139) or PTHrP (1-36), at 100 nM, to the cell cultures. Alkaline Phosphatase Activity Following incubations for only one 48 hour cycle as described above, cells were washed with phosphate-buffered saline (PBS) and cell extracts obtained with 0.1% Triton X-100. Alkaline phosphatase (ALP) activity was measured in cell extracts using p-nitrophenylphosphate as substrate, as described previously [10, 12]. Total RNA and Protein Extraction
Materials and Methods Reagents Human PTHrP (1-36) and human PTHrP (107-139) were kindly supplied by A. F. Stewart (Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, PA) and F. Rocal (Proteomics Unit, Centro Nacional de Biotecnologı´ a, Madrid), respectively. The analog [Asn10, Leu11, D-Trp12] PTHrP (7-34) amide (PTHrP [7-34]) was from Bachem (Bubendorf, Switzerland). Mouse monoclonal antibody against a-tubulin (T-S168) was obtained from Sigma (St. Louis, MO). Rabbit polyclonal antibodies against human osteoprotegerin (OPG, sc-11383) and receptor activator of nuclear factor-jB ligand (RANKL, sc-9073) were from
Cell total RNA and protein were isolated using guanidinium thiocyanate-phenol-chloroform extraction (Tri-Reagent; Molecular Research Center, Cincinnati, OH), according to the manufacturerÕs instructions. Cell extracts obtained with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) (buffer A), containing a phosphatase-inhibitor cocktail (Set II, Calbiochem), were used to analyze ERK phosphorylation. To obtain membrane protein extracts, cells were lysed with PBS/ 0.1% Triton X-100 and centrifuged at 2,000 · g for 10 minutes. The supernatant was then spun at 100,000 · g for 1 hour and the resulting pellet resuspended in buffer A (membrane protein extract). Protein was determined by a Coomassie reagentbased micromethod (Pierce, Rockford, IL), using bovine serum albumin as standard.
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Semiquantitative Reverse-Transcription Polymerase Chain Reaction
Electrophoretic Mobility Shift Assay
Cell total RNA (10 100 ng) was reverse-transcribed, and the resulting cDNA was amplified using a commercial kit (TitaniumTM One-step RT-PCR kit; Clontech, Palo Alto, CA) with the following specific primers: 5¢-CATGA GAGCCCTCACACTCC-3¢ (sense) and 5¢-CAGCAGAGCG ACACCCTAGACC-3¢ (antisense), corresponding to nucleotides 18-37 and 315-336, respectively, in the human OC gene (Genbank accession X51699) [31]; and 5¢-GGAATAG ATGTTACCCTGTG-3¢ (sense) and 5¢-TCAATGTCTTC TGCTCCC-3¢ (antisense), corresponding to nucleotides 680699 and 1015-1032, respectively, in the human OPG gene (Genbank accession U94332). These primers yield polymerase chain reaction (PCR) amplification products of 319 bp (OC) and 353 bp (OPG). Modified 18S primers (QuantumRNATM 18S Internal Standards; Ambion, Austin, TX) were used for 18S coamplification, as an internal control. This technology is based on the use of 18S rRNA primers and competimers modified at the 3¢ ends to block extension by DNA polymerase. This approach reduces the PCR amplification efficiency of 18S cDNA so that it can be in the same linear range as the target gene without affecting the performance of the latter, allowing relative quantification. The reaction mixture (10 lL) was incubated for 45 minutes at 48C and 2 minutes at 95C, followed by 32 35 cycles of 1 minute at 95C, 1 minute at 58 62C, and 2 minutes at 68C, with a final extension of 7 minutes at 68C. Preliminary experiments proved that these conditions provide submaximal amplification, corresponding to the linear segment in titration curves for reverse-transcription (RT) PCR amplification of each gene. PCR products were separated on 2% agarose gels, and bands were visualized by ethidium bromide staining and quantified by densitometric scanning. Densitometric values of PCR products were normalized against those of the corresponding 18S PCR product. Real-Time PCR Unlabeled human OPG, RANKL, and VEGF receptor 2 (VEGFR2) primers and TaqMan MGB probes were obtained by Assay-by-DesignTM (Applied Biosystems, Foster City, CA). cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (Promega) with random hexamer primers, and real-time PCR was carried out with an ABI PRISM 7500 system (Applied Biosystems), following the manufacturerÕs instructions. PCR amplification of 18S rRNA was done for each sample as sample loading control and to allow normalization between samples. mRNA copy numbers were calculated for each sample by using the cycle threshold value (‘‘arithmetic fit point analysis for the light cycler’’). Results were expressed in copy numbers, calculated relative to control cells, after normalization against 18S rRNA, as described [32]. Western Blot Analysis Cell membrane (50 lg protein) or total cell (20 60 lg protein) extracts were transferred onto nitrocellulose membranes (Amersham); blocked with 5% defatted milk in 100 mM Tris, 150 mM NaCl (pH 7.5), with 0.05% Tween-20; and then incubated overnight at 4C with primary polyclonal antibodies against RANKL (1:500 dilution), OPG (1:2,500 dilution), and pERK 1/2 or ERK 1/2 (each at 1:1,000 dilution) or with a monoclonal antibody against a-tubulin (1:10,000 dilution). After extensive washing, membranes were incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G (IgG) and developed by enhanced chemiluminescence (Amersham). The corresponding fluorogram bands were quantitated by densitometric scanning (ImageQuant; Molecular Dynamics, Sunnyvale, CA).
For assaying Runx2 activity, MG-63 cells were treated with PTHrP (107-139) at 100 nM for 3, 6, or 24 hours. Nuclear extracts were then prepared according to a commercially available procedure (NE-PER, Pierce), as previously described [30]. The double-stranded oligonucleotide 5¢-AGCTCCCAACCACATATCCT-3¢, containing a consensus sequence specific for Runx2 [33], was 5¢ end-labeled with 10 lCi [c32P]ATP and T4 polymerase. Nuclear extracts (3 lg protein) were incubated with 200,000 dpm of 32P-labeled oligonucleotide probe in 20 lL of a reaction mixture containing 10 mM Tris-HCl (pH 7.9), 100 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 4% glycerol, and 1 lg poly(dI-dC) for 20 minutes at 4C. Protein-DNA complexes were resolved on native 5% polyacrylamide/0.25·TBE gels. Gels then were dried and exposed to radiosensitive film. Statistical Analysis The data throughout the text are means ± standard error of the mean (SEM). Statistical analysis was performed by Kruskal-Wallis nonparametric analysis of variance followed by DunnÕs post-hoc test or Mann-Whitney test, as appropriate. P < 0.05 was considered significant. Results Effects of Transient and Continuous Exposure to PTHrP (107-139) on ALP and Runx2 Activities and OC mRNA in Human Osteoblast-like Cells
It has been previously reported that transient treatment with the N-terminal fragment of PTH or PTHrP, interacting with the common PTH1R in osteoblasts [34], stimulates various osteoblastic markers in several osteoblastic cell preparations [8, 9, 26 28]. We initially tested whether this type of exposure to PTHrP (1-36) reproduced this anabolic action in our cell culture system. We found that exposure to PTHrP (1-36), at 100 nM, for the first 24 hours of the 48-hour incubation period increased both ALP activity and OC mRNA expression in MG—63 cells (Fig. 1A and B). The same transient exposure regimen to PTHrP (107-139), at 100 nM, was also found to stimulate these osteoblastic markers in these cells (Fig. 1A and B). Moreover, we observed that 6-hour intermittent exposure to these peptides was efficient at stimulating OC gene expression in these cells (Fig. 1B). On the other hand, continuous exposure to these peptides abolished these osteoblastic differentiation markersÕ induction in these cells (Fig. 1A and B). A similar effect (about twofold over control) was observed for OC mRNA after transient (but not continuous) exposure to 100 nM PTHrP (107-139) in hOB cells. Consistent with the fact that OC contains several Runx2 binding sites [33], PTHrP (107-139), at 100 nM, induced a rapid increase in the activity of this transcription factor, which is critical for osteoblast maturation and bone formation [33], in MG-63 cells (Fig. 1C).
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Fig. 1. Effect of different exposure schedules to either PTHrP (107-139) (Ct) or PTHrP (1-36) (Nt) on ALP activity (A) and OC mRNA levels (B) in MG-63 cells. (A) Cells were exposed to each peptide (or vehicle, control [Co] cells), at 100 nM, for 24 hours and then incubated without the corresponding peptide for another 24-hour period or for 48 hours without medium change. Following incubations, ALP was measured in cell extracts. (B) Cells were exposed to each PTHrP peptide, at 100 nM, for the first 6 or 24 hours of each 48-hour incubation cycle and then cultured without the PTHrP peptide during the remainder of the cycle. This schedule was repeated three times. In the continuous exposure protocol, cells were exposed to each PTHrP peptide for 6 days. During this period, culture medium was changed every 48 hours. In control cells, the culture medium with vehicle was changed accordingly. The
autoradiograms show relative changes in OC mRNA and 18S mRNA (a constitutive control), which were coamplified by RT-PCR. Values are means ± SEM of at least three determinations in duplicate. *P < 0.05, **P < 0.01 vs. corresponding vehicle-treated control value (100%). (C) Time course of Runx2 activation induced by Ct peptide, at 100 nM, in MG-63 cells. Relative intensities of Runx2-DNA binding activity from two independent measurements are indicated at the top (fold-induction, means). As specificity controls, the retarded band in some nuclear extracts from Ct peptide-stimulated cells at 24 hours disappeared by preincubation with an excess (100·) of the unlabeled Runx2 consensus oligonucleotide but not by an unrelated NF-jB-binding oligonucleotide (not shown).
Effects of Transient and Continuous Exposure to PTHrP (107-139) on OPG and RANKL in Human Osteoblast-like Cells
the other hand, continuous exposure to this peptide reversed these changes in both OPG and RANKL in these cells (Figs. 2A and 3). PTHrP (107-139), at 100 nM, also induced OPG (mRNA and protein) expression in hOB cells (Figs. 2B and 4A). RANKL protein was undetected by Western analysis in hOB cell membranes. However, using real-time PCR, RANKL mRNA levels were low but detectable in hOB cells and PTHrP (107139) significantly inhibited expression of this gene in these cells (Fig. 2B).
OPG, a potent inhibitor of osteoclast differentiation and function by acting as a decoy receptor for RANKL, appears to increase with osteoblast differentiation [35, 36]. We found herein that OPG mRNA expression was significantly induced by intermittent exposure to PTHrP (107-139), at 100 nM, during MG-63 cell growth (Fig. 2A). Furthermore, OPG protein expression was increased to a similar magnitude by this pattern of exposure to this peptide within 10 100 nM in these cells (Fig. 3A). In contrast, using the same treatment schedule, PTHrP (107-139) within the same dose range decreased RANKL protein expression in MG-63 cell membrane extracts (Fig. 3B). Thus, transient exposure to PTHrP (107-139), at 100 nM, increased the OPG/ RANKL ratio by about threefold in MG-63 cells. On
Mechanism of Bone Anabolic Effects Induced by Transient Treatment with PTHrP (107-139) in Human Osteoblast-like Cells
Previous findings in other osteoblastic cell preparations support the existence of specific receptors for the Cterminal domain of PTHrP, different from PTH1R, in
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Fig. 2. Effect of different exposure schedules to PTHrP (107-139) (Ct) on OPG and RANKL mRNA expression in MG-63 (A) and hOB (B) cells. MG-63 cells were treated with this peptide, at 100 nM, as indicated in the legend to Figure 1B. hOB cells were transiently exposed to PTHrP (107-139), at 100 nM, for 24 hours (or 6 hours since the results were not significantly different from those at 24 hours) during three 48hour cycles, as described for MG-63 cells. OPG and RANKL mRNA levels were measured by real-time PCR. Autoradiogram shows changes in OPG and 18S mRNA coamplified by RT-PCR (A, left). Relative changes in OPG and RANKL gene expression are means ± SEM from three to five independent measurements. *P < 0.05, **P < 0.01 vs. corresponding vehicletreated control (Co) value. A.U., arbitrary units.
osteoblasts [11, 23]. We found herein that cell preincubation with 1 lM PTHrP (7-34), a PTH1R antagonist [37], blocked the stimulatory effect on OPG protein expression triggered by transient exposure to PTHrP (136) but not that induced by PTHrP (107-139) in both hOB and MG-63 cells (Fig. 4A and B). Various in vitro and in vivo studies support the concept that IGF-I mediates the bone anabolic effects of the Nterminal domain common to PTH and PTHrP [17, 18, 26, 27]. We examined the putative role of IGF-I on the osteogenic action of PTHrP (107-139) in MG-63 cells. As expected, we found that addition of an anti-IGF-I antibody, but not preimmune rabbit serum, abrogated OC mRNA and OPG protein induction by intermittent exposure to PTHrP (1-36) in these cells (Fig. 5A and B). Interestingly, this antibody was inefficient at inhibiting the overexpression of these osteoblastic products after transient exposure to PTHrP (107-139) (Fig. 5). VEGF is now viewed as an emerging local osteogenic factor [24]. Consistent with our previous findings [25], transient exposure to each PTHrP peptide similarly increased VEGF mRNA levels in MG-63 cells (not shown), whereas only PTHrP (107-139) increased the
gene expression of VEGFR2 (Fig. 6A). Furthermore, this overexpression was also observed in hOB cells (Fig. 7C). In addition, the specific VEGFR2 tyrosine kinase inhibitor SU5614, at 1 lM, abolished the stimulatory effect of PTHrP (107-139) on ALP activity in MG-63 cells. Thus, the corresponding values were (nmol/min/mg protein): 5.5 ± 0.8 in control cells and 5.4 ± 0.9 or 8.4 ± 1.5 in PTHrP (107-139)-treated cells in the presence or absence of SU5614, respectively (P < 0.05 between the latter value and either control or SU5614-treated value, n = 4), whereas this inhibitor failed to affect ALP activity in control cells (not shown). Moreover, SU5614 also abrogated the gene expression of OC and OPG in MG-63 and hOB cells (Fig. 6B and C). Several lines of evidence indicate that protein kinase C (PKC) activation is a major pathway involved in the osteoblastic effects of PTHrP (107-139) [10 12, 23, 25, 30]. In the present study, either BIM (25 nM) or calphostin C (200 nM), two PKC inhibitors [10 12, 23, 30], abolished the stimulatory effect of this PTHrP peptide on both VEGFR2 and OPG mRNA expression in MG-63 and hOB cells (Fig. 7A and C). Previous
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Fig. 3. Effect of intermittent or continuous exposure to PTHrP (107-139) (Ct) on OPG and RANKL protein expression in MG-63 cells. Cells were treated with this peptide, at 100 nM, or at different doses (M) (A and B, left), as indicated in the legend to Figure 1B. Total cell protein extracts (A) or cell membrane extracts (B) were analyzed by Western immunoblotting using polyclonal anti-OPG or anti-RANKL antibodies, detecting a single band corresponding to an apparent molecular weight of 55 or 42 kDa, respectively. Protein loading was similar in each well, as assessed by a-tubulin (A) or Ponceau S staining (a stained band corresponding to an apparent molecular weight of 47 kDa is shown) (B) as internal controls. Relative intensities of corresponding OPG and RANKL signals from four independent measurements are indicated at the top (fold-induction, means).
studies have shown that ERK activation might occur downstream of PKC activation in osteogenic cells from rat bone marrow and in MG-63 cells [8, 9]. We found herein that pERK was rapidly (within 15 minutes) stimulated by 100 nM PTHrP (107-139) in the latter cells (Fig. 7B). This stimulation was unaffected by SU5614 but abolished by both calphostin C and PD098059 (10 lM), an inhibitor of ERK phosphorylation [8, 9] (Fig. 7B). Furthermore, the latter inhibitor abrogated the effect of PTHrP (107-139) on the expression of both VEGFR2 and OPG mRNA in MG-63 and hOB cells (Fig. 7A and C). Neither of the aforementioned inhibitors significantly affected the nonstimulated control gene expression of these osteoblastic products or ERK phosphorylation in these cells (not shown). Discussion
In the present study, we report that intermittent, but not continuous, exposure to the C-terminal PTHrP domain, containing the osteostatin epitope, induces several
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Fig. 4. Effect of PTHrP (7-34) on OPG protein induction by transient exposure to either PTHrP (107-139) (Ct) or PTHrP (1-36) (Nt) in hOB (A) and MG-63 (B) cells. Cells were treated with each PTHrP peptide (at 100 nM) or vehicle (control, Co) for 24 hours, as indicated in the text, in the presence or absence of 1 lM PTHrP (7-34). Total cell protein extracts were analyzed by Western immunoblotting using a polyclonal antiOPG antibody. Protein loading in each well was assessed by atubulin. Relative intensities of OPG signals from three independent measurements are indicated at the top (fold-induction, means).
osteoblast differentiation features in vitro in human osteoblast-like cells. The present data also support the novel notion that this action of PTHrP (107-139) occurs by VEGFR2 activation in these cells. Whether the present in vitro findings might translate into an anabolic effect of this PTHrP peptide in bone awaits further studies in vivo. Only limited and inconsistent data are currently available from such studies. In one of these studies, a daily dose (3 nmol/100 g) of PTHrP (107-111) given for 13 days to ovariectomized adult rats increased femoral bone mass and calcium content to values similar to those in sham-operated control animals [15]. Surprisingly, however, this treatment with osteostatin decreased osteoid surface and increased bone resorption (associated with thinner trabeculae and higher osteoclast number) in cancellous bone measured at the distal epiphysis of the femur [15]. In another study, more consistent with earlier in vitro observations [20], a marked decrease in bone resorption indexes was induced by local daily injection of PTHrP (107-139) (in the range pM-nM) into adult mouse calvariae [19]. In the latter report, a much smaller reduction in some indexes of bone formation, accompanied by an upward trend to an increase in mineralized bone area, was observed in peptide-treated calvariae [19]. Thus, at
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Fig. 5. Effect of neutralizing anti-IGF-I antibody on OC mRNA (A) and OPG protein (B) expression induced by intermittent exposure to either PTHrP (107-139) (Ct) or PTHrP (1-36) (Nt) in MG-63 cells. Cells were treated with each PTHrP peptide (at 100 nM) or vehicle (control, Co) for 24 hours, as indicated in the text, in the presence of an anti-IGF-I antibody or an equivalent dilution of preimmune rabbit serum. (A) The autoradiogram shows changes in OC and 18S mRNA, a constitutive control, coamplified by RT-PCR. (B) Total cell protein extracts were analyzed by Western immunoblotting using a polyclonal anti-OPG antibody. Protein loading was similar in each well, as assessed by a-tubulin. Relative intensities of OC/18S mRNA ratio and OPG signals from three independent measurements are indicated at the top (foldinduction, means).
least under some experimental conditions, a positive effect of C-terminal PTHrP on bone formation might result from its inhibitory effect on bone resorption, considering the well-known coupling of bone formation and bone resorption. Of interest in this regard, a preliminary report has recently shown that mice with knockin deletion of the mid- and C-terminal PTHrP domains develop osteoporosis within 2 3 weeks of age. This was apparently due to decreased osteoblastic bone formation in both trabecular and cortical bone associated with decreased expression of Runx2, ALP, and OC [21]. Previous in vitro studies by us and other investigators, using several rat and human osteoblastic cell preparations, demonstrate that PTHrP (107-139) can directly interact with osteoblasts [10 12, 22, 23, 25, 30]. In one of these studies, this PTHrP peptide was found to inhibit ALP activity in growing rat osteoblastic osteosarcoma UMR-106 cells [12]. Meanwhile, PTHrP (107-139), at ‡0.1 nM, also reduced ALP activity and C-terminal type I procollagen secretion, but did not affect OC secretion, in confluent primary cultures of hOB cells [10]. These previously reported findings are actually consistent with the present data since the aforementioned effects were observed after 3 4 days of exposure to the C-terminal PTHrP peptide, which mimics its continuous addition herein. On the other hand, a recent report has shown
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Fig. 6. (A) Effect of intermittent exposure to either PTHrP (107-139) (Ct) or PTHrP (1-36) (Nt) on VEGFR2 mRNA expression in MG-63 cells. Cells were treated with each PTHrP peptide, at 100 nM, or vehicle for 24 hours, as indicated in the text. Effect of the VEGFR2 signaling inhibitor SU5614 on OPG and OC induction by transient exposure for 24 hours to Ct peptide in MG-63 (B) and hOB (C) cells. The autoradiograms show relative changes (indicated at the top; foldinduction, means) in OPG and OC mRNA and 18S mRNA, a constitutive control, coamplified by RT-PCR (B). Relative changes in VEGFR2 (A) and OPG (C) mRNA levels, measured by real-time PCR, are means ± SEM from three independent measurements in duplicate. *P < 0.05 vs. corresponding vehicle-treated control (Co, A, C) and SU5614treated (C) values. A.U., arbitrary units.
that shorter (6 24 hours) exposure to PTHrP (107-139), between 0.1 and 10 nM, induced a moderate but statistically significant increase in cell growth in subconfluent osteoblastic cells from fetal rat calvariae [22]. Collectively, these findings and the present results in various osteoblastic cell systems suggest that diverse effects of the C-terminal PTHrP domain can occur, depending on the temporal pattern of its exposure in osteoblasts. The putative mechanisms involved in the differential response of osteoblastic cells to transient or continuous exposure to PTH are still unclear. Interestingly, the developmental temporal variance of anabolic and catabolic responses to this hormone seems to be related to osteoblastic maturation, associated with changes in
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Fig. 7. Effect of different inhibitors on VEGFR2 and OPG induction by transient exposure to PTHrP (107-139) (Ct) in MG-63 (A) and hOB (C) cells. Cells were treated with Ct peptide, at 100 nM, or vehicle for 24 hours, in the presence or absence of the different inhibitors, as indicated in the text. The autoradiograms show relative changes (indicated at the top; foldinduction, means) in OPG and 18S mRNA, a constitutive control, coamplified by RT-PCR (A, left). Relative changes in VEGFR2 (A and C, right) and OPG (C, left) mRNA levels, measured by real-time PCR, are means ± SEM from three independent measurements in duplicate. *P < 0.05 vs. corresponding vehicle-treated control value (Co) and inhibitortreated values. A.U., arbitrary units. (B) Effect of Ct peptide, at 100 nM, in the presence or absence of different inhibitors, on ERK activation in MG-63 cells. Western blot analysis was performed in cell extracts using antibodies against pERK 1/2 or ERK 1/2. Co, nonstimulated control (at 15 minutes). Relative densitometric values corresponding to pERK/ERK changes are means ± SEM from three independent measurements in duplicate. *P < 0.05 vs. corresponding vehicle-treated Co value (100%) and calphostin C (Calph)- or PD098059-treated values.
PTH1R [26, 28]. We found herein that the stimulatory effect on OPG induced by transient treatment with PTHrP (1-36), but not with PTHrP (107-139), was abolished by PTHrP (7-34), a PTH1R antagonist [37], in both hOB and MG-63 cells. This finding lends support to the existence of a receptor for PTHrP (107-139) different from PTH1R in osteoblasts, as previously suggested [11, 23]. Putative changes in the expression of such a specific receptor for this peptide and/or signaling might explain the different responses associated with diverse exposure times to PTHrP (107-139) in hOB cells. This hypothesis should be tested in further studies. It is now considered that IGF-I has an important role in the anabolic actions of PTH in bone [17, 26, 27]. In the present study, the stimulatory effects of intermittent exposure to PTHrP (1-36) on both OC and OPG
expression were abrogated by a neutralizing IGF-I antibody in MG-63 cells. This is consistent with the results of an early report showing that IGF-I neutralization could prevent PTHrP-stimulated collagen synthesis in cultured fetal rat calvariae [18]. On the other hand, we found that the stimulation triggered by transient exposure to PTHrP (107-139) on the aforementioned osteoblastic products was not affected by the IGF-I antibody in MG-63 cells. Thus, IGF-I does not appear to be required for this effect of PTHrP (107-139) in these cells. However, a specific and permeable inhibitor of VEGFR2 was found to abolish the effects of PTHrP (107-139) on ALP activity and on both OC and OPG mRNA expression in human osteoblastic cells. VEGF, a potent angiogenic factor, appears to have an important
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role in the well-characterized coupling of angiogenesis and bone formation [24, 38]. Moreover, VEGF might act as a mediator for well-known osteogenic factors, such as vitamin D3 and PTH [39], and it has been shown to upregulate OPG expression in endothelial cells [40]. In addition, a previous report using a mouse preosteoblastic cell line suggested a regulatory role of VEGFR2 on osteoblast differentiation [41]. In this way, in a mouse model of osteoporosis associated with hyperthyroidism, thyroid-stimulating hormone inhibited osteoblast differentiation by downregulating VEGFR2 signaling [42]. In fact, the putative use of VEGF for the treatment of osteoporosis has been suggested by a recent study in rabbits showing that adenovirus-mediated VEGF gene transfer increased osteoblast activity and bone formation [43]. Taken together, the present findings and our previous data [25] support the notion that PTHrP (107-139) may stimulate osteoblast differentiation, at least in part, through modulation of theVEGF/ VEGFR2 system. In the present study, we found that stimulation of OPG and VEGFR2 overexpression by intermittent exposure to PTHrP (107-139) was apparently dependent on PKC activation in hOB cells. In this regard, activation of PKC has been shown to play a critical role in the modulation of OPG and VEGFR2 expression in human osteosarcoma SaOS-2 and endothelial cells, respectively [44, 45]. In addition, these effects induced by PTHrP (107-139) were antagonized by an ERK activation inhibitor in human osteoblastic cells. In this regard, another report has shown that PTH1R signaling via several G protein-coupled pathways, including PKC activation, can apparently converge in mitogen-activated protein kinase activation to increase MG-63 cell differentiation [8]. Our present data suggest that interaction of PTHrP (107-139) with its specific receptor may trigger a similar intracellular pathway as a mediator of at least part of its effects in human osteoblastic cells. In conclusion, this study demonstrates that different patterns of exposure to PTHrP (107-139) in vitro can exert biphasic responses in human osteoblastic cells, affecting their differentiation. The present results provide new evidence supporting a potential anabolic action of this PTHrP domain through the VEGF/VEGFR2 system in osteoblasts.
Farmace´uticos (Bilbao, Spain). A. R. de G. and V. A. are fellows of Fundacio´n Conchita Ra´bago. A. R. de G. was the recipient of a travel stipend from SEIOMM to attend the American Society for Bone and Mineral Research meeting in Seattle (2004). M.V.A-A. is the recipient of a research contract from Institute Carlos III.
Acknowledgment. We are indebted to I. Torres, PhD (Instituto Ramo´n y Cajal, Madrid), and N. Vilaboa, PhD (Laboratorio de Metabolismo O´seo, Hospital Universitario La Paz, Madrid), for supplying the anti-IGF-I antibody and hOB cell cultures, respectively. This work was supported in part by grants from the Instituto de Salud Carlos III (C03/08, PI050363, and PI050117), Ministerio de Educacio´n y Ciencia of Spain (SAF2005 05254), and Comunidad Auto´noma de Madrid (GR/SAL/0417/2004) and by two awards, from the Sociedad Espan˜ola de Investigaciones O´seas y Metabolismo Mineral (SEIOMM)-Italfa´rmaco (Alcobendas, Spain) and SEIOMM-Fundacio´n Espan˜ola de Productos Quı´ micos y
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