Abstract. The N-terminal region of parathyroid hormone (PTH)and
PTH-related protein (PTHrP) interacts with a common PTH/PTHrPreceptor in
osteoblasts. These cells synthesize PTHrP, but itsrole in bone turnover is
unclear. Intermittent treatment withN-terminal PTHrP or PTH stimulates bone
growth in vivo, possiblyby increasing local bone factors. In
addition, C-terminal PTHrP(107-139), which does not bind to the PTH/PTHrP
receptor, appearsto affect bone resorption in vivo and in
vitro, although itseffect on bone formation in vivo remains
controversial. Boneangiogenesis is an often overlooked but critical event in
theprocess of bone remodeling. Recently, PTH (1-34) has been shownto induce
gene expression of vascular endothelial growth factor(VEGF), a potent
angiogenic factor, by osteoblastic cells. However,no data are available on
the effect of PTHrP (107-139) on VEGFexpression in these cells. Using
semiquantitative reverse transcriptionfollowed by PCR, we found that PTHrP
(107-139), between 10 nMand 1 pM, increased VEGF mRNA in human osteoblastic
(hOB) cellsfrom trabecular bone. This effect of this agonist, at 10 nM,was
maximal (fivefold for VEGF165, and twofold for VEGF121,
comparedto control) within 1 to 4 h. This effect was similar to thatinduced
by PTHrP (1-34) in these cells, as well as in humanosteosarcoma MG-63 cells,
using Northern blot analysis. Moreover,the effect of both peptides, added
together at 100 pM, was nothigher than that observed with each peptide alone
in hOB cells.The effects of PTHrP (107-139) and that of PTHrP (1-34) were
abolishedby actinomycin D in hOB cells. In these cells, the protein kinaseC
inhibitor staurosporine, but not the protein kinase A inhibitorH89, inhibited
the increase in VEGF mRNA induced by 10 nM PTHrP(107-139). PTHrP (107-139),
at 10 nM, also stimulated cytosolicVEGF immunostaining in hOB cells, and VEGF
secretion into themedium conditioned by hOB or MG-63 cells for 24 h, which
was(ng/mg protein): 10 ± 1 or 5 ± 3 (control), respectively,
and21 ± 1 or 11 ± 2 (PTHrP [107-139]-stimulated),respectively.
Furthermore, medium conditioned by these cellsfor 24 h in the presence of 10
nM PTHrP (107-139), with or without10 nM PTHrP (1-34), increased about 30%
bovine aortic endothelialcell (BAEC) growth at 48 h. This effect was
inhibited by addinga specific anti-VEGF antibody to the BAEC incubation
medium.These findings demonstrate that the C-terminal domain of PTHrPinduces
expression and secretion of VEGF, a main angiogenicfactor, in hOB cells and
MG-63 cells. This relationship betweenPTHrP and VEGF has potential
implications for both bone vascularizationand bone formation, and
neoangiogenesis in PTHrP-producing tumors.
The important role of bone microvasculature in osteogenesiswas postulated
decades ago (1). In fact,
electrical stimulationinduces an increase of bone formation associated with a
parallelincrease of blood capillaries
(2). On the other hand, a
reductionin blood flow and number of sinusoids is a well known featureof
osteoporotic bone, and impairment of vascular supply leadsto bone necrosis
(3,4,5).
It is now known that vascularizationoccurs before osteogenesis during both
intramembranous and endochondralbone formation. During the latter process,
bone-forming osteoblastsline the terminal capillary wall of the sprouting
capillariesinvading the epiphyseal plate, so that cartilage matrix in this
areais degraded by osteoclasts, and then replaced by new bone aftervascular
invasion
(6,7).
In fracture repair, callus formationdepends on the restoration of blood
supply in the fracture cavity.Endothelial cells in the vicinity of the
fracture gap becometransformed, associated with the appearance of
dedifferentiatedmesenchymal cells and new osteoblasts
(8). Moreover, endothelial
cellsare known to synthesize various bone formation inducers
(9,10).
Takentogether, these findings support the point of view that bonegrowth and
angiogenesis occur in a coordinate manner. However,the mechanisms of
interaction between bone endothelium and osteoblasticcells to promote an
adequate angiogenesis to encompass boneformation are still unclear.
Vascular endothelial growth factor (VEGF) is a potent and specificmitogen
for endothelial cells, which has a key role in normaland pathologic
angiogenesis (11). Alternative
mRNA splicingyields four different VEGF molecular species of 206, 189, 165,
and121 amino acids (12). The
latter two isoforms are diffusibleproteins detected in the majority of cells
expressing the VEGFgene (12).
A recent study has demonstrated that VEGF mRNA ispresent in hypertrophic
chondrocytes in the mouse epiphysealgrowth plate, where VEGF-dependent blood
vessel invasion appearsto be essential for coupling cartilage resorption with
boneformation (13). Recently,
mRNA for VEGF and its receptors, flt-1and KDR, have been detected in
preosteoblasts and osteoblastsin the human fracture callus
(14). VEGF mRNA is rapidly
inducedby prostaglandin E1 (PGE1) and PGE2,
potent stimulators of boneformation in vivo, in rat osteoblastic
cells (15). Moreover,insulin
growth factor I and 1,25-dihydroxyvitamin D3
(1,25(OH)2D3),two bone anabolic factors, stimulate VEGF
expression in humanosteoblastic (hOB) cells
(10,16,17).
Parathyroid hormone (PTH)-related protein (PTHrP) and the commontype 1
PTH/PTHrP receptor are present in a variety of normaland fetal tissues and
cell types, including chondrocytes andosteoblasts in skeletal tissue
(18). Both PTH and PTHrP, when
administeredin an intermittent manner in vivo, are anabolic in bone
(19,20).
Thiseffect is likely to be accounted for by their action on thesynthesis of
various growth factors in bone cells
(21,22).
Inthis regard, PTH (1-34) has recently been shown to increaseVEGF gene
expression or protein secretion, alone or in the presenceof
1,25(OH)2D3, respectively, in human osteosarcoma SaOS-2
cells
(16,17).
Furthermore,the bone anabolic effects of PTHrP might not be restricted toits
N-terminal domain. Thus, PTHrP peptides containing the 107-111epitope, so
called osteostatin, have been shown to inhibit boneresorption in rodents both
in vitro and in vivo
(20,23,24,25).
Inaddition, local injection of low doses of one of these peptidesto mouse
calvaria decreases the number of osteoblasts
(23),while intermittent
administration of high doses of PTHrP (107-111)into ovariectomized rats
decreases trabecular bone formation,but restores cortical bone mass
(20). In these in
vivo models,however, a putative positive effect of C-terminal PTHrP on
bonemass might be difficult to assess in the presence of the marked
inhibitionof bone resorption induced by this PTHrP domain
(20,23,25).
Recentin vitro studies from our group and from other investigators
(26,27,28)
havefound either inhibitory or stimulatory effects of PTHrP (107-139),a
putative PTHrP fragment (29),
on osteoblastic proliferationand/or differentiation, depending on the type of
osteoblasticcells studied. Thus, the true effect of this functional domainof
PTHrP on bone formation is unclear.
In the present study, we specifically investigated the effectof the
C-terminal PTHrP peptide PTHrP (107-139) on VEGF expressionat the
transcriptional and protein levels in hOB cells. We havealso assessed the
putative intracellular mechanism involvedin the effect of this PTHrP peptide
on VEGF expression in thesecells. The effect of PTHrP (107-139) on VEGF
expression wascompared with that of the PTH-like peptide PTHrP (1-34) in hOB
cells.
Materials
Human PTHrP (1-34) amide (PTHrP [1-34]), human PTHrP (107-139),actinomycin
D, and staurosporine were from Sigma (St. Louis,MO).
N-[2-((p-bromocinnamyl)amino)-ethyl]-5-isoquinolinesulfonamiden
dihydrochloride(H89) was from Calbiochem (San Diego, CA). VEGF cDNA probe was
kindlydonated by Dr. B. Williams (Leicester University, United Kingdom).This
probe corresponds to a domain within exon 3 in the VEGFgene, a coding region
common to the four VEGF isoforms.
Cell Cultures
hOB cells were cultured from trabecular bone explants obtainedat the time
of surgery on osteoarthritic patients. The patients(six women and three men,
ages 56 to 81 yr) had no evidenceof metabolic bone disorders. The bone
fragments were culturedin Dulbecco's modified Eagle's medium (DMEM)
containing 15%fetal bovine serum (FBS), 100 IU/ml penicillin, and 100
µg/mlstreptomycin in 5% CO2 at 37°C, as described
(26). Experimentswere
performed with these cells subcultured at first passageand grown to
confluence, which display features of functionalosteoblasts
(26). The hOB cells were
preincubated for 48 h inphenol red-free DMEM (1 g/L glucose) supplemented
with 10 nMvitamin K, 50 µg/ml ascorbic acid, 0.1% bovine serum albumin
(BSA),and antibiotics (differentiation medium), and then the testagents,
dissolved in the same medium, were added for varioustime periods.
Human osteosarcoma cells MG-63 (American Type Culture CollectionCRL 1427,
Manassas, VA) were grown in DMEM with 10% FBS andantibiotics. Confluent cells
were FBS-depleted for 24 h beforestimulation with the agonists for various
time periods.
Bovine endothelial cells (BAEC), obtained by digestion withtype II
collagenase, were cultured in minimum essential mediumwith D-valine,
supplemented with 20% FBS, iron, nonessentialamino acids, and antibiotics, as
described (30). Cells were
usedbetween two to four passages.
Assay of BAEC Proliferation
BAEC grown to 60 to 70% confluence in the aforementioned mediumwere
incubated for 24 h in hOB differentiation medium, replacing0.1% BSA by 1%
FBS. Then, the conditioned medium from hOB cellswas added for 48 h. Cell
number was counted using a Neubauerchamber (Afora, Madrid, Spain). In some
experiments, a neutralizingconcentration (2 µg/ml) of a specific
anti-human VEGFmonoclonal antibody (Sigma)
(30), or the same
concentrationof nonimmunogenic IgG, was added to BAEC.
Isolation of Total RNA and Reverse Transcription-PCR
VEGF mRNA levels in hOB cells were assessed by reverse transcription
followedby PCR (RT-PCR). Total RNA was extracted by guanidinium
thiocyanate-phenol-chloroformextraction
(31). Total RNA aliquots were
added to a reactionmixture (10 µl) with 1 mM MgSO4, 0.2 mM of
each deoxynucleotidetriphosphate, 1 U of avian myeloblastosis virus reverse
transcriptase,1 U of thermostable DNA polymerase from Thermus flavus
(AccessRT-PCR System; Promega, Madison, WI), and 0.5 µM of thespecific
primers for the human VEGF gene
(32):
5'-CATGAACTTTCTGCTGTCTTGG-3'(sense), and
5'-CTCACCGCCTCGGCTTGTCAC-3' (antisense).
RT-PCR of mRNA encoding the 121, 165, 189, and 206 amino acidVEGF
isoforms, which arise by alternative splicing, yields 459-,591-, 663-, and
714-bp products, respectively. The restrictionenzyme sites for EcoRI
and BamHI were introduced in the 5' positionof the primer
sequence to facilitate sequencing of PCR products.The housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was coamplified, using
specific primers for the humangene
(33) as a constitutive
control.
Total RNA and the primers were preincubated for 5 min at 65°C.Then,
the reaction mixture was incubated for 45 min at 48°C,and 2 min at
95°C, followed by 30 to 35 cycles of 1 minat 95°C, 1 min at 60°C,
and 2 min at 68°C, witha final extension of 7 min at 68°C. The PCR
products wereseparated on 2% agarose gels, and bands were visualized by
ethidiumbromide staining and quantified by densitometric scanning
(ImageQuant;Molecular Dynamics, Sunnyvale, CA). Densitometric values forVEGF
PCR product were normalized by comparison with the GAPDHproduct signal. The
identity of the PCR products were confirmedby automatic sequencing in a 373
DNA Sequencer (Applied Biosystems,United Kingdom).
Northern Blot Analysis
Because availability of hOB cell cultures was limited, and theirRNA yield
was poor, we further analyzed the response of VEGFmRNA to PTHrP fragments in
MG-63 cells, which express both VEGFand the PTH/PTHrP receptor
(16,34).
Total RNA was isolated fromMG-63 cells as described above. Twenty micrograms
of total RNAwere size-fractionated on 1% agarose gel containing 1.2 M
formaldehydeand transferred to nylon membranes (Hybond-N+, Amersham,
Buckinghamshire,United Kingdom). The membranes were prehybridized at 42°C
for8 h, and hybridized overnight at 42°C with 106 cpm/ml
32P-labeledhuman VEGF cDNA probe. This probe was labeled with
[-32P]dCTPusing a random-primed DNA labeling kit
(Boehringer Mannheim,Mannheim, Germany). Filters were washed for 30 min at
42°Cin 1x saline-sodium phosphate-ethyl-enediaminotetra-acetic
acid,0.3% sodium dodecyl sulfate. Filters were then exposed on KodakX-Omat
film at -70°C for 4 to 6 d. The filters were reprobedwith a 28S cDNA
probe.
VEGF Enzyme-Linked Immunoassay
hOB cells and MG-63 cells were incubated in phenol red-freemedium with
supplements and FBS-depleted DMEM, respectively,in the presence or absence of
the test agents for 24 h. Theconditioned medium was collected, and kept at
-20°C forup to 3 wk before assay. VEGF was determined by a sandwich-type
enzyme-linkedimmuno-assay (ELISA) (CYTElisaTM VEGF assay;
CYTImmune Sciences,Inc., College Park, MD) combining a capture monoclonal
antibodyprecoated onto the microplate, and a signal biotinylated polyclonal
antibody.The assay sensitivity is 5 pg/ml, and detects both secretedVEGF
isoforms. Cell protein was assayed in 0.1N NaOH-solubilizedcell extracts,
using the Bradford method
(35). Data were expressedas
the amount of VEGF secreted per milligram of cell protein.
Immunocytochemistry
hOB cells were incubated with the agonists in phenol red-freemedium with
supplements on chamber slides (Nalge Nunc International,Naperville, IL) for
24 h. The cells were then fixed in ethanol.Immunocytochemistry was performed
using an affinity-purifiedanti-VEGF IgG. This antibody was raised by rabbit
immunizationwith a peptide corresponding to a common region for all human
VEGFisoforms, coupled to keyhole limpet hemocyanin
(36). The cellswere incubated
with this affinity-purified antibody (10 µg/ml)for 2 h at room
temperature, followed by sequential additionof avidin-biotin-peroxidase
complex and 3,3'-diaminobenzidine(Sigma)
(27). As negative controls,
some cell preparations wereincubated with 10 µg/ml nonimmunogenic rabbit
IgG.
Statistical Analyses
Results are expressed as means ± SD. Statistical analyseswere
performed by unpaired t test or one-way ANOVA, when appropriate.
P< 0.05 was considered significant.
Agonist-induced changes of VEGF expression were evaluated byusing
semiquantitative RT-PCR. We carried out preliminary titrationexperiments to
determine the suitable amount of total RNA andthe number of cycles that
provide submaximal amplification ofVEGF in our RT-PCR system. We found that
30 to 35 cycles and10 ng of total RNA from either PTHrP-stimulated or
nonstimulatedhOB cells fit this requirement. Using these conditions, PTHrP
(107-139),at 10 nM, was found to induce an approximately fivefold increaseof
VEGF165 mRNA within 1 to 4 h in these cells, declining thereafter
(Figure 1, A and C).Moreover,
this PTHrP peptide, at 10 nM, also significantlyincreased by approximately
two-fold VEGF121 mRNA in hOB cellswithin the same time period
(Figure 1, A and C). Because a
recentstudy in human osteosarcoma SaOS-2 cells has shown that PTH(1-34)
stimulates VEGF gene expression at 48 h
(16), we thensought to
examine the effect of PTHrP (1-34), interacting witha common PTH/PTHrP
receptor in osteoblasts (37),
on VEGF mRNAin hOB cells. We found that this peptide, similar to PTHrP
(107-139),at 10 nM increased both VEGF165 and VEGF121
mRNA in these cells(Figure 1, A and
C). This effect of either PTHrP (107-139) orPTHrP (1-34), both at
10 nM, on VEGF mRNA was of a range similarto that induced by 10% FBS, a
positive control (11), in
thesecells (Figure 1A). The
stimulatory effect of PTHrP (107-139)or PTHrP (1-34) on the expression of
each VEGF isoform in hOBcells was already detectable with 1 pM of these
peptides (notshown), and it was neither additive nor synergistic when both
peptideswere added together at a submaximal concentration (100 pM)
(Figure 1B).Using Northern
blot analysis, we have also found that eachPTHrP peptide stimulated VEGF mRNA
in MG-63 cells (Figure 2).
Figure 1. (A) Time-dependent effect of parathyroid hormone (PTH)-related protein
(PTHrP) (107-139) or PTHrP (1-34) on vascular endothelial growth factor (VEGF)
mRNA in human osteoblastic (hOB) cells. The cells were stimulated in fetal
bovine serum (FBS)-free medium with supplements, as described in Materials and
Methods, with or without either peptide at 10 nM for one-half to 24 h, or 10%
FBS for 2 h. (B) Effect of PTHrP (107-139) (C) and PTHrP (1-34) (N), alone or
together, at 100 pM, on VEGF mRNA in these cells for 2 h. These are
representative of three different patients. (C) Changes in VEGF121
and VEGF165 mRNA levels, corrected to those of GAPDH mRNA, in hOB
cells after stimulation with either PTHrP (107-139) or PTHrP (1-34), at 10 nM,
for 2 h. Data are mean ± SD corresponding to five different patients.
P < 0.025, between values corresponding to every VEGF isoform,
each PTHrP peptide, and nonstimulated control (100%).
Figure 2. Northern blot analysis of VEGF mRNA from MG-63 cells treated with or
without either PTHrP (1-34) or PTHrP (107-139) at 10 nM for 3 h. Twenty
micrograms of total RNA was loaded in each lane. The filters were hybridized
with 32P-labeled 204-bp human VEGF cDNA and 28S ribosomal probes,
and then were exposed to autoradiographic film.
Additional studies were carried out to further characterizethe mechanism
involved in the upregulation of VEGF mRNA by bothPTHrP (107-139) and PTHrP
(1-34) in hOB cells. To examine whetherthe increased VEGF mRNA induced by
both PTHrP peptides was theresult of an increased transcription, we incubated
the hOB cellswith the RNA polymerase inhibitor actinomycin D
(38) at 10 µg/mlin the
presence and absence of these PTHrP peptides. We foundthat actinomycin D
inhibited VEGF mRNA in nonstimulated hOBcells, and abolished the stimulatory
effect of 10 nM PTHrP (107-139)or PTHrP (1-34) on VEGF mRNA in these cells
(Figure 3). Meanwhile,
actinomycinD did not affect the mRNA of the housekeeping gene GAPDH inthese
cells (Figure 3).
Figure 3. Effect of different agents on VEGF mRNA induced by PTHrP (107-139) and
PTHrP (1-34) in hOB cells. The cells were treated in FBS-free medium and
supplements with each PTHrP peptide at 10 nM in the presence or absence of
different agents for 2 h. Actinomycin D (ActD) was added at 10 µg/ml,
immediately before addition of PTHrP peptides. Staurosporine (100 nM) and H89
(100 nM) were added 1 h before stimulation with each PTHrP peptide. These are
representative of two different patients.
Previous studies have suggested the involvement of protein kinaseC (PKC)
in various effects of PTHrP (107-139) on cell proliferationand/or
differentiation in a variety of cell types, includinghOB cells
(24,26,27,38,39,40,41).
In the present study, 100nM staurosporine, a PKC inhibitor
(42), in contrast to 100 nM
H89,a PKA inhibitor (43),
abrogated the effect of 10 nM PTHrP (107-139)on VEGF mRNA in hOB cells
(Figure 3). Conversely, 100 nM
H89abolished the effect of 10 nM PTHrP (1-34) on VEGF expressionin these
cells (Figure 3).
We next assessed whether PTHrP (107-139) increased VEGF proteinin hOB
cells. A weak VEGF immunostaining was found in the cytoplasmof nonstimulated
hOB cells (Figure 4A).
Treatment with 10 nMPTHrP (107-139) for 24 h increased VEGF positivity in
thesecells (Figure 4B). In
contrast, no cell staining was observedafter incubation with nonimmunogenic
IgG (Figure 4C). PTHrP
(107-139),at 10 nM, was also found to increase VEGF levels in hOB
cell-conditionedmedium. Thus, at 24 h, immunoreactive VEGF in this medium was
(ng/mgprotein): 10 ± 1 (nonstimulated hOB cells) and 21 ±1
(PTHrP [107-139]-treated hOB cells) (n = 3; P < 0.01).At
this time period, 10 nM PTHrP (107-139) also induced an increaseof VEGF
secretion in MG-63 cells, which was (ng/mg protein):5 ± 3 and 11
± 2 in cells untreated or treatedwith 10 nM PTHrP (107-139),
respectively (n = 3; P < 0.05).Furthermore, the medium
conditioned by hOB cells for 24 h inthe presence of 10 nM PTHrP (107-139)
induced a significantincrease in BAEC growth, compared to that of these cells
incubatedin hOB cell-conditioned medium without this peptide
(Figure 5).This effect was
abolished by the simultaneous addition ofan anti-VEGF antibody, but not by
nonimmunogenic IgG (Figure 5).This effect of the conditioned medium from PTHrP (107-139)-treatedhOB cells
was not further increased by the simultaneous presenceof various
concentrations (100 pM to 10 nM) of PTHrP (1-34)(not shown).
Figure 4. Immunocytochemical staining for VEGF in hOB cells. The cells were incubated
without (A) or with (B) 10 nM PTHrP (107-139) for 24 h. Cells were incubated
with either primary anti-VEGF antibody (A and B) or nonimmunogenic rabbit IgG
(C). This is representative of results in three different patients.
Figure 5. Effect of the hOB cell-conditioned medium (CM) after stimulation with or
without 10 nM PTHrP (107-139) for 24 h on bovine aortic endothelial cell
(BAEC) growth after 48 h in the presence or absence of 2 µg/ml anti-VEGF
IgG or nonimmunogenic IgG. Results are mean ± SD of values from two
different hOB cell cultures tested on two different BAEC cultures, performed
at least in triplicate. *,**P < 0.01,
compared with untreated CM, with or without nonimmunogenic IgG.
aP < 0.01, compared with the corresponding anti-VEGF
value.
Angiogenesis is known to play a significant role in bone formation.Current
data support the existence of a finely regulated cross-talkbetween
endothelium and bone cells during bone growth and repair
(6,7,8).
However,the interactions between endothelial cells and other cells inthe
bone microenvironment are poorly understood. A variety ofagents that
stimulate bone formation, including PTH (1-34),induce expression and
secretion of VEGF, a potent angiogenicfactor, in osteoblastic cells
(10,15,16,17).
On the other hand,dexamethasone, which induces avascular bone necrosis
(44), inhibitsVEGF expression
in rat calvaria-derived osteoblastic cells andhuman osteosarcoma SaOS-2 cells
(15,17).
Recent findings indicatethat suppression of blood vessel invasion by systemic
administrationof a VEGF receptor chimeric protein to mice leads to an
inhibitionof cartilage resorption and an impaired bone formation
(13).These data point to VEGF
as a critical coordinator of cartilageremodeling and ossification in
endochondral bone formation.
In the present study, PTHrP (107-139) was found to increaseVEGF mRNA in
hOB cells and MG-63 cells. This induction was rapidand transient in hOB
cells, and appears to occur primarily atthe transcriptional level, consistent
with previous findingsin hOB and rat osteoblastic cells after stimulation
with PGE2and 1,25(OH)2D3, respectively
(15,16).
Moreover, the potencyof the effect triggered by PTHrP (107-139) on VEGF
expressionwas of the same range as that induced by
1,25(OH)2D3 or 10%FBS in hOB cells (reference
16 and present results,
respectively),suggesting its putative physiologic importance. The enhanced
VEGFmRNA after stimulation with PTHrP (107-139) was accompaniedby an
increase in VEGF protein in these cells, as shown by anincreased VEGF
immunostaining in hOB cells and a stimulatedimmunoreactive VEGF in the
conditioned medium from PTHrP (107-139)-treatedhOB and MG-63 cells. In
addition, stimulation of hOB cells withPTHrP (107-139) also increased
biologically active VEGF in thesecells' conditioned medium, as demonstrated
by its inductionof an anti-VEGF antibody-inhibitable BAEC growth
stimulation.
The effects of 1,25(OH)2D3 and PGE2 on
VEGF expression in osteoblasticcells appear to depend on PKC and PKA
activation, respectively,consistent with the hypothesis that both kinases are
involvedin the induction of VEGF increase
(45). In the present study,
usingspecific inhibitors, we found that VEGF mRNA induction by PTHrP
(107-139)in hOB cells is likely to occur by activation of PKC. This is
consistentwith our previous observation that PTHrP (107-139) fails to
stimulatePKA activity in rat osteoblastic osteosarcoma UMR 106 and hOBcells
(26,27).
On the other hand, this PTHrP peptide has beenshown to increase PKC activity
in rat osteoblastic osteosarcomacells ROS 17/2.8
(39), and also in rat spleen
lymphocytes andhuman keratinocytes
(40,41).
A previous report, using Northern blot analysis, has shown astimulatory
effect of PTH (1-34) on VEGF mRNA in human osteosarcomaSaOS-2 cells
(16). We found herein that
PTHrP (1-34), similarto PTHrP (107-139), increased VEGF mRNA in hOB and MG-63
cells.In addition, our results suggest that this effect is cAMP-dependent,
supportingthe hypothesis that cAMP stimulation is the major pathway involved
inthe bone anabolic effects of the N-terminal region of PTH andPTHrP
(46,47).
Both PTHrP (107-139) and PTHrP (1-34), added together at a concentration
thattriggers a submaximal effect on VEGF expression, failed to inducea
greater increase of VEGF mRNA compared to that triggered byeach peptide alone
in hOB cells. This suggests the existenceof a common final effector pathway
affecting VEGF expression,and supports that a cross-talk in the signal
transduction pathwaysassociated with the response to each PTHrP peptide
occurs inthese cells, as noted previously
(38,48).
The physiologic relevance of our findings is supported by thefact that
several PTHrP fragments, including the domains 1-36and 107-139, are secreted
by a variety of cells
(29,49).
Inaddition, C-terminal PTHrP fragments containing its 107-111region
accumulate in plasma of uremic patients, associated withthe decrease of renal
function (50). The latter
PTHrP domainhas been shown to induce either a somewhat inhibitory or a
stimulatoryeffect on bone formation in mouse calvaria or ovariectomizedrats,
respectively, associated with a dramatic inhibition ofbone resorption in both
in vivo models
(20,23).
Therefore, thesestudies do not rule out a possible direct effect of this
PTHrPregion on bone formation. In this regard, we recently foundthat PTHrP
(107-139) increases the expression of interleukin-6,a putative osteoblast
differentiation factor, in hOB cells
(38,51).
Ourpresent findings support a role for PTHrP (107-139) as a boneformation
promoter. In addition, considering the aforementionedinhibitory effect of
this peptide on bone resorption, our dataare also consistent with the
hypothesis that bone angiogenesiscan occur independently of bone resorption,
as recently suggested
(52).
In summary, the results herein support that the C-terminal domainof PTHrP,
in a manner similar to that of its PTH-like region,could promote bone
angiogenesis by inducing VEGF in normal andtransformed human osteoblastic
cells. Additional studies arerequired to clarify the functional significance
of the cross-talkbetween the different intracellular pathways that appeared
tobe linked to the effect of both PTHrP regions on VEGF expressionin hOB
cells. However, it is reasonable to speculate that thismight provide
alternative pathways to ensure bone vascularization.Our findings also suggest
a putative mechanism that might beresponsible, at least in part, for
neoangiogenesis in PTHrP-producingtumors.
Acknowledgments
This work was supported in part by grants from Fondo de
InvestigaciónSanitaria (96/1167, 97/307,
97/0341, and 00/0125), ComunidadAutónoma de
Madrid (CAM P7/083/0004), and Fundación
Españolade Productos
Químicos y
Farmacéuticos, S.A. ofSpain. Drs. De Miguel
and Alvarez-Arroyo are postdoctoral researcherssupported by CAM. Olga Martin
is a fellow of CAM (no. 07/016/96).We thank Dr. R. Bragado (Immunology
Department, Fundación
JiménezDíaz)
for providing us with the affinity-purified anti-VEGFantibody used for the
immunocytochemical studies. We also thankDr. B. Williams (Leicester
University, United Kingdom) for thedonation of the VEGF cDNA probe.
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Received for publication July 16, 1999.
Accepted for publication November 2, 1999.
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Top
Abstract
Introduction
-32P]dCTP using a random-primed DNA labeling kit
(Boehringer Mannheim, Mannheim, Germany). Filters were washed for 30 min at
42°C in 1x saline-sodium phosphate-ethyl-enediaminotetra-acetic
acid, 0.3% sodium dodecyl sulfate. Filters were then exposed on Kodak X-Omat
film at -70°C for 4 to 6 d. The filters were reprobed with a 28S cDNA
probe. 
