PGE2 and BMP-2 in bone and cartilage metabolism: 2 intertwining pathways

Abstract

Osteoarthritis and lesions to cartilage tissue are diseases that frequently result in impaired joint function and patient disability. The treatment of osteoarthritis, along with local bone defects and systemic skeletal diseases, remains a significant clinical challenge for orthopaedic surgeons. Several bone morphogenetic proteins (BMPs) are known to have osteoinductive effects, whereof BMP-2 and BMP-7 are already approved for clinical applications. There is growing evidence that the metabolism of bone as well as the cartilage damage associated with the above disease processes are strongly inter-related with the interactions of the inflammation-related pathways (in particular prostaglandin E2 (PGE2)) and osteogenesis (in particular bone morphogenetic protein-2 (BMP-2)). There is strong evidence that the pathways of prostaglandins and bone morphogenetic proteins are intertwined, and they have recently come into focus in several experimental and clinical studies. This paper focuses on PGE2 and BMP-2 intertwining pathways in bone and cartilage metabolism, and summarizes the recent experimental and clinical data.

Full text

REVIEW / SYNTHÈSE
PGE
2
and BMP-2 in bone and cartilage
metabolism: 2 intertwining pathways
Marcel Haversath, Isabelle Catelas, Xinning Li, Tjark Tassemeier,
and Marcus Jäger
Abstract: Osteoarthritis and lesions to cartilage tissue are diseases that frequently result in impaired joint function and
patient disability. The treatment of osteoarthritis, along with local bone defects and systemic skeletal diseases, remains a
significant clinical challenge for orthopaedic surgeons. Several bone morphogenetic proteins (BMPs) are known to have
osteoinductive effects, whereof BMP-2 and BMP-7 are already approved for clinical applications. There is growing
evidence that the metabolism of bone as well as the cartilage damage associated with the above disease processes are
strongly inter-related with the interactions of the inflammation-related pathways (in particular prostaglandin E
2
(PGE
2
)) and
osteogenesis (in particular bone morphogenetic protein-2 (BMP-2)). There is strong evidence that the pathways of
prostaglandins and bone morphogenetic proteins are intertwined, and they have recently come into focus in several
experimental and clinical studies. This paper focuses on PGE
2
and BMP-2 intertwining pathways in bone and cartilage
metabolism, and summarizes the recent experimental and clinical data.
Key words: osteoblasts, osteoclasts, prostaglandins, bone morphogenetic proteins, cellular signaling.
Received 11 November 2011. Accepted 28 May 2012. Published at www.nrcresearchpress.com/cjpp on 7 November 2012.
Abbreviations: ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; ALP, alkaline phosphatase; BMP,
bone morphogenetic proteins; BMP-2, bone morphogenetic protein-2; BMPR-Ia (ALK3), type Ia bone morphogenetic receptor;
BMPR-Ib (ALK6), type Ib bone morphogenetic receptor; BMPR-II, type II bone morphogenetic receptor; cAMP, cyclic
adenosine monophosphate; cFMS, colony-stimulating factor-1 receptor (c-fms proto-oncogene product); c-FOS, a transcription
factor encoded by the FOS gene, a proto-oncogen; COX-2, cyclooxygenase-2; CSF-1, colony stimulating factor-1; Dlx5, a
transcription factor encoded by the Dlx5 gene; EP1, prostaglandin E receptor 1 encoded by the PTGER1 gene; EP2,
prostaglandin E receptor 2 encoded by the PTGER2 gene; EP3, prostaglandin E receptor 3 encoded by the PTGER3 gene; EP4,
prostaglandin E receptor 4 encoded by the PTGER4 gene; ERK, extracellular-signal regulated kinase; ER-␣, estrogen receptor
␣; FDA, US Food and Drug Administration; Fra-1, FOS-related antigen 1, a transcription factor belonging to the c-FOS gene
family; Fra-2, FOS-related antigen 2, a transcription factor belonging to the c-FOS gene family; HO, heterotopic ossifications;
IL-1␣, interleukin-1␣; IL-6, interleukin-6; IL-8, interleukin-8; IP3, inositol triphosphate; JNK, c-Jun N-terminal kinases,
belonging to the mitogen-activated protein kinases family; MAPK, mitogen-activated protein kinases, responsive to extracel-
lular stimuli; MMP-1, matrix metalloproteinase-1; MMP-13, matrix metalloproteinase-13; MMP-9, matrix metalloprotei-
nase-9; Msx2, a transcription factor encoded by the msh homeobox 2 gene; NF-␬B, nuclear factor ␬-light-chain-enhancer of
activated B cells; OA, osteoarthritis; OCN, osteocalcin; ONO-4891, a selective prostaglandin E receptor 4 activator; OPG,
osteoprotegerin; OPN, osteopontin; Osx, osterix transcription factor, required for the expression of osteopontin; PG, prosta-
glandins: PGE2, prostaglandin E2: PGES, prostaglandin E synthase: PGHS-2, prostaglandin H synthase type 2; PKA, protein
kinase A; PLA2, phospholipase A2; PLC, phospholipase C; PTH, parathormone; RANK, receptor activator of nuclear factor
␬B; RANKL, receptor activator of nuclear factor-␬B ligand; rhBMP-2, recombinant human bone morphogenetic protein-2;
R-SMAD, receptor regulated SMAD (a transcription factor family that transduce TGF-␤ signals); Runx2, runt-related
transcription factor-2 (associated with osteoblastic differentiation); TGF-␤, transforming growth factor-␤; TNF-␣, tumor
necrosis factor-␣; TRAF6, TNF receptor associated factor-6 (a protein that transduces TNF signals); Vit. D, vitamin D.
M. Haversath, T. Tassemeier, and M. Jäger. Orthopaedic Department, University Hospital, University of Duisburg-Essen,
Hufelandstrasse 55, D-45147 Essen, Germany.
I. Catelas. Department of Mechanical Engineering, Department of Surgery, and Department of Biochemistry, Microbiology and
Immunology, University of Ottawa, Ottawa, ON K1N 6N5, Canada; Department of Mechanical Engineering, University of Ottawa, 161
Louis Pasteur A-206, Ottawa, ON K1N 6N5, Canada.
X. Li. Department of Orthopaedic Surgery, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
Corresponding author: Marcus Jäger (e-mail: marcus.jaeger@uk-essen.de).
1434
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Résumé : L’ostéoarthrite et les lésions du cartilage sont des maladies qui résultent fréquemment en une réduction de la fonction
articulaire et en une invalidité du patient. Le traitement de l’ostéoarthrite qui s’accompagne d’anomalies osseuses locales et de
maladies squelettiques systémiques pose toujours un défi clinique significatif aux chirurgiens orthopédiques. Plusieurs protéines
morphogènes de l’os, les BMP, sont connues pour exercer des effets ostéoinducteurs, parmi lesquelles BMP-2 et BMP-6 sont
déja
`
approuvées aux fins d’applications cliniques. Ilyadeplus en plus de preuves que le métabolisme de l’os et le dommage au
cartilage associé aux maladies mentionnées plus haut sont fortement interreliés aux interactions des voies de l’inflammation
(notamment la voie de la prostaglandine E
2
, PGE
2
) et de l’ostéogenèse (notamment la voie de la bone morphogenetic protein-2,
BMP-2). Il existe une preuve solide que les voies des prostaglandines et des BMP s’entrecroisent, et elles ont été l’objet d’une
attention particulière dans plusieurs études expérimentales et cliniques. Cet article se concentre sur l’entrecroisement des voies de
la PGE
2
et de la BMP-2 dans le métabolisme de l’os et du cartilage, et résume les dernières données expérimentales et cliniques.
Mots-clés : ostéoblastes, ostéoclastes, prostaglandines, protéines morphogènes de l’os, signalisation cellulaire.
[Traduit par la Rédaction]
Relevance of prostaglandins and bone morphogenetic
proteins in orthopaedics
Prostaglandins (PG) play an important role in bone forma-
tion, inflammation processes, and perfusion. They are lipid,
arachidonic acid-derived mediators that are produced by a
variety of cells and act in an autocrine and paracrine fashion to
maintain local homeostasis. PGE
2
is one of the most investi-
gated prostanoids and is also known to be crucially involved in
regulating bone formation associated with fracture healing and
heterotopic ossification on the one hand, and bone resorption
associated with inflammation and osteolytic effects of meta-
static cancer on the other (Blackwell et al. 2010).
Bone morphogenetic proteins (BMPs) are multi-functional
growth factors belonging to the transforming growth factor ␤
(TGF
␤
) gene superfamily. There are about 30 different mol-
ecules within this group of mediators. BMPs play an important
role in mesoderm formation. Thus, besides their role in the
formation of cartilage and bone, they are also involved in the
development and maintenance of various other mesodermal
tissues, including kidneys and blood vessels (Chen et al.
2004). In the post-natal skeleton, BMPs are produced by the
periosteal cells and mesenchymal cells of the marrow stroma.
Three of them, i.e., BMP-2, -4, and -7, have been reported to
induce de novo bone formation in vitro and in vivo (Sampath
et al. 1990). Recent research has revealed that other BMPs
such as BMP-6 and BMP-9 are also osteoinductive, and might
have potential for future clinical applications (Kamiya 2011;
Luther et al. 2011).
This review focuses on PGE
2
and BMP-2 as 2 crucial medi-
ators involved in the pathogenesis and therapeutic treatment of
orthopaedic diseases. In the first section, the main functions of
both mediators in the pathogenesis of orthopaedic diseases are
highlighted. In a second section, the biochemical intertwining
pathways of PGE
2
and BMP-2 are illustrated.
PGE
2
and BMP-2: relevant mediators in bone formation
and repair
PGE
2
, as a subtype, is known to be one of the most impor-
tant local regulators of bone metabolism. Within bone, PGE
2
is primarily produced by osteoblasts. Enhanced production of
PGE
2
by local osteoblasts can be found adjacent to skeletal
injury. In this inflammatory environment, PGE
2
is essential for
new bone formation and bone healing (the effects of PGE
2
on
osteoblasts in the presence of inflammation are illustrated in
Fig. 1). For example, O’Keefe et al. (2006) showed that local
delivery of PGE
2
enhanced bone formation at the cortical bone
graft junction. Other studies have reported that mice lacking
cyclooxygenase 2 (COX-2), and thus unable to produce PGE
2
,
as well as aged mice with reduced COX-2 expression showed
significant delayed bone healing after injury (Zhang et al.
2002; Naik et al. 2009). PGE
2
activates osteoblasts directly
and osteoclasts indirectly through interactions with osteoblasts
via the RANK signaling pathway (Boyle et al. 2003). Four
different G-protein-coupled PGE
2
receptors have been identi-
fied: EP1, EP2, EP3, and EP4. All of these were detected in
osteoblasts of different animal species (Narumiya et al. 1999).
However, in human osteoblasts, only EP3 and EP4 were
observed. Activation of EP4 has been shown to rescue im-
paired bone fracture healing in COX-2
⫺/⫺
mice (Xie et al.
2009). Similarly, Naik et al. (2009) have shown that COX-2/EP4
agonists may compensate for deficient molecular signals that result in
the reduced fracture healing associated with aging.
Activation of EP2 and EP4 leads to a stimulation of adenylate
cyclase and increased cAMP levels as a second messenger sys-
tem. On the other hand, EP3 receptor activation results in lower
cAMP levels. High cAMP levels are linked to anabolic bone
remodelling (Hakeda et al. 1986). Therefore, EP2 and EP4
receptors seem to play a crucial role in bone remodelling.
Furthermore, selective stimulation of EP2 receptors of osteo-
blasts leads to differentiation of osteoblasts (Choudhary et al.
2008), which in turn cause differentiation of osteoclasts via the
RANK/RANKL signaling pathway. With regards to EP1 re-
ceptor, there is evidence that its activation inhibits osteo-
blast differentiation (Zhang et al. 2011). The EP1 receptor
activates phospholipase C followed by induction of calcium
mobilization.
PGE
2
production is also increased through the upregulation
of COX-2 expression via an autogenous stimulation mecha-
nism (Suda et al. 1998). In addition, selective stimulation of
EP4 receptors of osteoblasts leads to differentiation of osteo-
blasts and upregulation of COX-2 expression, but less than
that of a single EP2 activation (Sakuma et al. 2004). Overall,
PGE
2
has been shown to have anabolic effects on bone re-
modelling in vivo (Graham et al. 2009). However, severe side
effects such as diarrhea, lethargy, and flushing preclude PGE
2
as a therapeutic agent in bone diseases. In recent animal experi-
ments using selective EP2 and EP4 receptor agonists, minimal
side effects were seen when compared with using PGE
2
alone
(Paralkar et al. 2003). Therefore, selective agonists of EP2 or
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Fig. 1. Intertwining pathways in the osteoblasts. Skeletal injury leads to local production and release of PGE
2
and BMP-2. These mediators
activate specific osteoblast receptors followed by intracellular signaling via different pathways. There is evidence that both PGE
2
and BMP-
2 act via the MAPK signaling pathway, which ultimately leads to osteopontin, alkaline phosphatase, and osteocalcin expression. Since
PGE
2
and BMP-2 signaling are still not fully understood, an “unknown gene (X)” has been added to the figure, as future studies may
reveal the contribution of an additional gene.
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EP4 may have a therapeutic potential for enhancing bone
formation and bone healing.
BMPs are some of the most investigated cytokine molecules
that are critically involved in osteoblast differentiation and bone
formation. Specifically, BMP-2, -4, -6, -7, and -9 are known to be
osteoinductive by inducing the differentiation of mesenchymal
stem cells into osteoblast precursors, and promoting the matura-
tion of osteoblasts through the increase of osteoblast differentia-
tion gene expression. Recent research also suggests that BMP-2
in particular, directly promotes not only the differentiation of
osteoblasts but also osteoclasts (Jensen et al. 2010).
BMP-2 activates osteoblasts directly via 2 types of trans-
membrane receptors: BMPR-I and BMPR-II, as illustrated in
Fig. 1. BMPR-I is further subclassified into BMPR-IA (ALK3)
and BMPR-IB (ALK6). All receptors possess intrinsic serine/
threonine kinase activity. Activation leads to phosphorylation
of different specific intracellular signal molecules including
so-called R-SMADs. Subsequent activation of transcriptional
factors (i.e., Dlx5 and Runx2) leads to enhanced expression of
osteoblast differentiation marker genes, resulting in new bone
formation (Ryoo et al. 2006). The anabolic effect of BMP-2
for bone regeneration in vivo is used in clinical practice today:
recombinant BMP-2 (rhBMP-2, Infuse
®
Bone Graft, Medtronic
Sofamor Danek, Inc., Memphis, Tennessee; or dibotermin alfa as
InductOs
®
, Wyeth Pharmaceuticals, Berkshire, UK) is locally
applied in cases of impaired bone healing. Figure 2 illustrates
clinical results obtained after treatment of a severe symptom-
atic acetabular osteolysis with cancellous bone graft supple-
mented with BMP-2. It has received approval by FDA for
specific indications in open tibial fractures, anterior single-
level lumbar spinal fusion, and certain oral maxillofacial and
dental regeneration applications.
PGE
2
and BMP-2 in fracture repair
Bone fracture leads to local hypoxia due to disruption of the
vasculature. Hypoxia is then followed by the release of PGE
2
from osteoblastic cells (Lee et al. 2010). Thus, a physiological
increase of endogenous, local PGE
2
-production can be quan
-
titatively measured after fracture. In a tibial diaphyseal frac-
ture model, PGE
2
administration restored cartilage formation
and significantly reduced the fracture gap in Fra-1 transgenic
mice, which are normally incapable of initiating callus forma-
tion at the fracture site (Yamaguchi et al. 2009). PGE
2
has
been reported to enhance fracture healing in murine experi-
ments, mainly through activation of the EP4 receptor (Xie et
al. 2009). Similarly, Marui et al. (2006) demonstrated accel-
erated bone healing and decreased incidence of sternal wound
complications after median sternotomy in diabetic rats with
local application of a PGE
2
EP4 receptor-selective agonist. By
comparison with selective EP4 receptor activation, selective
EP2 receptor activation leads to increased COX-2 expression
(Sakuma et al. 2004). COX-2 is known to enhance bone
healing in vivo (Xie et al. 2009). Xie et al. showed that
administration of EP4 receptor-selective agonists can rescue
impaired fracture healing in COX-2
⫺/⫺
knockout mice, but not
EP2-selective agonists (Xie et al. 2009). Although both acti-
vated receptors are followed by cAMP-elevation, they seem to
play different roles during bone repair. Xie et al. also mea-
Fig. 2. Computerized tomography (CT scan) and X-ray pictures of a 73-year-old patient with a severe and symptomatic acetabular osteolysis
beyond the acetabular component of a hip arthroplasty, owing to wear particles (white arrow), and after up to 4 years of follow-up. At the time of
revision, cancellous bone graft supplemented with BMP-2 was added after curettage of the defect and application of bone-marrow cells. (a) The
critical size defect healed within 12 weeks, as demonstrated by the CT scans. (b) The images show that BMP-2 can lead to significant new bone
formation within an inflammatory tissue. The latest follow-up showed solid implant integration over 4 years (white double-arrow). The patient is
free of pain, has an unlimited walking distance, and can return to previous sport activities (such as bike riding).
Haversath et al. 1437
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sured increased levels of matrix metalloproteinase-9 (MMP-9)
after targeted stimulation of EP4-receptors but not EP2 recep-
tors. MMP-9 is known to induce angiogenesis during endo-
chondral ossification. Hence, these results demonstrate that
EP2 and EP4 might in part operate via different signaling
pathways.
Hypoxia not only induces PGE
2
release but also enhances
BMP-2 expression in osteoblasts (Tseng et al. 2010). BMPs
can be found along collagen fibers of human bone matrix.
After fracture, BMPs diffuse from bone matrix and stimulate
osteoprogenitor cells, further enhancing BMP production. There
is evidence that BMP-2 is required for fracture repair. Indeed,
mice lacking the ability to produce BMP-2 showed spontaneous
fractures due to inferior bone strength, and these fractures do not
heal with time. Interestingly, however, nearly normal skeletal
development could be found in these animals (Tsuji et al. 2006).
A prospective, randomized, controlled, single-blind study in-
volving 450 patients with an open tibial fracture was published
in 2002, showing that locally administered rhBMP-2 at the
fracture site significantly reduces the frequency of secondary
interventions, accelerates fracture and wound-healing, and re-
duces the infection rate (Govender et al. 2002). This study was
the basis for pre-market approval by the FDA of rhBMP-2 for
open tibial fractures. In a recently published, evidence-based
Cochrane review, 11 randomized controlled trials and 4 eco-
nomic evaluations were analyzed to assess the effectiveness of
clinically used rhBMP (i.e., rhBMP-2 and rh-BMP-7) for fracture
healing in adults (Garrison et al. 2010). The review ascertained
the paucity of data on the use of BMP in fracture healing.
Furthermore, the heavily industry-sponsored scientific involvement
in currently available evidence-based studies was criticized. Gar-
rison et al. (2010) stated that there is limited evidence for BMP
being more effective than controls for acute tibial fracture
healing. Economical assessment suggested that the use of
BMP is only favourable in patients with the most severe
fractures.
PGE
2
and BMP-2 in osteoporosis
Osteoporosis is a disease characterized by bone loss result-
ing in increased fracture risk. The disease is subdivided into 2
types: post-menopausal (type I), and senile (type II) osteopo-
rosis. An imbalance of bone formation and bone resorption is
considered to play the main role in pathophysiology of both
types of osteoporosis.
Bone remodelling is a well-balanced, constant process of
bone resorption by osteoclasts and bone formation by osteo-
blasts. In elderly post-menopausal women, the extent of re-
sorption is higher than formation, leading to bone loss. The
excess of resorption is linked to low levels of serum estrogen,
such as 17␤-estradiol, known to induce osteoprotegerin in
osteoblasts, and which inhibits RANKL-mediated osteoclast
activation resulting in decreased bone resorption (Hofbauer
and Heufelder 2001). Some studies have shown that bone loss
can be suppressed by administration of PGE
2
in vivo (Li et al.
1995; Ke et al. 1998). For example, Li et al. (1995) revealed
that systemic PGE
2
treatment can prevent bone loss in orchi
-
dectomized rats. In an ovariectomized rat model with estab-
lished osteopenia, Ke et al. (1998) demonstrated that systemic
PGE
2
treatment completely restored maximum load and stiff
-
ness of bone after 30 days of treatment. In addition, a signif-
icant increase in maximum load and stiffness in both rapidly
growing and adult male rats could be observed in non-
orchidectomized animals. Yoshida et al. (2002) reported that
restoration of bone mass was primarily mediated via the PGE
2
EP4 receptor. Indeed, using a murine model, the authors
showed that only EP4-deficient mice (⫺/⫺) did not show de
novo bone formation after PGE
2
treatment. In mice lacking the
EP1, EP2, or EP3 receptor, massive formation of woven bone
was histologically identified after treatment. However, animal
studies using PGE
2
in vivo have reported severe side effects
that include severe diarrhea, hair loss, and decreased physical
activity, precluding the use of PGE
2
as a therapeutic agent for
osteoporosis in humans (Graham et al. 2009). Interestingly,
further investigations have demonstrated that ONO-4891, a
selective EP4 agonist, prevented bone loss and restored bone
mass and strength in ovariectomized rats (Yoshida et al. 2002).
In contrast to PGE
2
, no severe side effects at the dose required
for bone formation were observed when the EP4 agonist was
utilized. Thus, selective EP4 agonists may represent promising
therapeutic agents for osteoporosis (Yoshida et al. 2002).
BMPs play an important role in the pathophysiology of
osteoporosis. In fact, Urist, who discovered BMPs in 1965,
labelled osteoporosis as “a bone morphogenetic protein auto-
immune disorder” in 1985. In a rat model, Bessho and Iizuka
(1993) found an age-dependent decreasing activity of BMPs
after implantation of purified BMP into the calf muscles.
These results were supported by Fleet et al. (1996), who
reported that aging impairs rhBMP-2-induced bone formation
in rats, and explained their results by a reduced number of
mesenchymal stem cells associated with aging or a change in
the responsiveness of these target cells to rhBMP-2. Turgeman
et al. (2002) showed that bone mass could be recovered by
rhBMP-2 when administered systemically to osteopenic mice.
This effect was coupled with an increased number of adult
mesenchymal stem cells in bone. Recent research also dem-
onstrated that 17␤-estradiol, which is known to be lowered in
type I osteoporosis, promotes the induction of BMP-2 in mouse
mesenchymal stem cells, mainly via the activation of estrogen
receptor alpha (ER-␣)(Zhou et al. 2003). In opposition to
17␤-estradiol, glucocorticoids are known to induce osteopenia,
particularly as a result of long-term pharmacological intake. Luppen
et al. (2008) revealed that expression of BMP-2 is inhibited in
glucocorticoid-arrested osteoblasts, and that rhBMP-2 restores bone
mass mainly via activation of R-SMAD signaling molecules.
PGE
2
and BMP-2 in local osteolysis
Periprosthetic osteolysis is a common cause of aseptic loos-
ening in total joint arthroplasty. Wear particles in peripros-
thetic tissues stimulate the production of inflammatory
cytokines such as TNF-␣, PGE
2
, and IL-6 (Bukata et al. 2004).
In particular, titanium wear particles were found to activate
PGE
2
production in fibroblasts through a COX-2 dependent
pathway (Bukata et al. 2004). Other investigations also dem-
onstrated a pro-inflammatory response of human osteoblasts to
cobalt ions, leading to increased secretion of chemokines
including PGE
2
(Queally et al. 2009). A decrease in the
secretion of alkaline phosphatase and in calcium deposition,
markers of osteoblastic differentiation, was measured. Other
studies demonstrated that PGE
2
could lead to the activation of
periprosthetic fibroblasts through EP4, followed by an ele-
vated expression of RANKL, which is known as the final
effector of osteoclastogenesis and bone resorption (Tsutsumi
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et al. 2009). Aseptic loosening due to osteolysis can therefore,
at least in part, be initiated by local PGE
2
production resulting
in a subsequent indirect activation of osteoclasts predomi-
nantly and short-handed periprosthetic osteoblasts. Taken to-
gether, one must consider that besides the role of PGE
2
in
indirect activation of osteoclasts via osteoblasts or fibroblasts,
other cytokines such as TNF-␣, IL-1␣, IL-6, and IL-8, which
are produced by a variety of cells, can also lead to osteoclast
activation. Hence, periprosthetic osteolysis is a complex, in-
flammatory process mediated by different cytokines, including
PGE
2
, which collectively result in an excess of osteoclastic
activity.
rhBMP-2 has been reported to lead not only to the differ-
entiation of osteoblasts, but also of osteoclasts (Majid et al.
2010). In recent studies using rhBMP-2 for spinal lumbar
interbody fusions, several adverse effects were discovered,
including vertebral osteolysis, which is known to be a self-
limiting event (Rihn et al. 2009). Rihn et al. discovered ver-
tebral osteolysis in 5 out of 86 patients who underwent single-
level transforaminal interbody fusion (TLIF) in combination
with rhBMP-2. However, 2 cases of ectopic bone formation
were also described (Rihn et al. 2009). Although the precise
pathophysiology of rhBMP-2-induced vertebral osteolysis re-
mains unclear, there are hints that this complication may be a
dose-dependent phenomenon. The current literature suggests
that the carrier used to apply rhBMP-2 may play an important
role in the development of vertebral osteolysis. Different stud-
ies have proposed that scaffolds providing more sustained and
localized delivery would be advantageous for decreasing the
rate of systemic and local complications such as vertebral
osteolysis (Xu et al. 2009). The dose–response relationship
between locally released rhBMP-2 and the extent of osteoclas-
togenesis remains controversial (Kaneko et al. 2000; Toth et
al. 2009). In a sheep model, Toth et al. reported that increasing
local rhBMP-2 levels lead to enhanced osteoclastic resorption
of peri-implant bone. However, this osteoclastic effect was
transient, and was followed by progressive bone healing and
bone formation (Toth et al. 2009). BMPR-I and BMPR-II
receptors were detected in osteoblasts as well as osteoclasts,
and current literature suggests that BMP-2 activates osteoclasts
directly (Kaneko et al. 2000; Jensen et al. 2010) and indirectly
through osteoclastic-promoting factors such as RANKL pro-
duced by osteoblasts or stromal cells (Abe et al. 2000). Recent
in vitro studies have revealed that BMP-2 leads to direct enhanced
activation of osteoclasts via SMAD signaling (P-SMAD1/5/8)
(Jensen et al. 2010). However, enhanced expression of
osteoclast differentiation genes by BMP-2 was RANKL-
dependent. In the absence of RANKL, BMP-2 was not able to
induce differentiation (Jensen et al. 2010). These findings were
supported by Itoh et al. (2001), suggesting that BMP-2-
induced enhancement of osteoclast differentiation was caused
by cross-communication between BMP receptor-mediated sig-
nals and RANK-mediated signals. However, they assumed
that the MAPK pathway rather than the SMAD pathway was
involved in osteoclast differentiation by BMP-2.
PGE
2
and BMP-2 in ectopic bone formation (heterotopic
ossifications)
Injury to soft tissue near bone can induce heterotopic ossi-
fications (HO) that are a common problem after orthopaedic
surgery. Using a rabbit model, Bartlett et al. (2006) found that
PGE
2
was required for the development of ectopic bone.
Non-steroidal anti-inflammatory drugs (NSAID) have been
given prophylactically to patients, resulting in a lower inci-
dence of HO. For example, Rapuano et al. (2008) found that
COX-2 inhibitors significantly reduced PGE
2
levels at the site
of the injury. Bartlett et al. (2006) reported on locally elevated
levels of different prostaglandins, including PGE
2
, prior to and
during the development of HO after muscle trauma in a rabbit
model. Within only 24 h after injury, they measured enhanced
PGE
2
levels at the site of the injury, and suggested that HO
prophylaxis with NSAIDs or prostaglandin receptor antago-
nists should be started immediately in patients undergoing
orthopaedic surgery. Furthermore, they demonstrated that the
induction of HO was mainly mediated via the PGE
2
EP2
receptor, and that elevated cAMP levels were critical in this
process. After administration of the EP1 and EP2 receptor
antagonist AH 6809, no development of HO could be found in
the presence of PGE
2
. These findings were partly supported by
Nakagawa et al. (2007) showing that ONO-4819 alone, a se-
lective agonist for the EP4 prostanoid receptor, was not capa-
ble of inducing alkaline phosphatase, which is an early
marker of osteoblast differentiation. However, in the pres-
ence of rhBMP-2, some studies demonstrated that systemic
and local administration of ONO-4819 enhanced ectopic bone
formation both in vivo and in vitro (Toyoda et al. 2005;
Nakagawa et al. 2007). Toyoda et al. assumed that ONO-4819
and BMP-2 had cooperative anabolic effects on bone metab-
olism, and that administration of ONO-4819 together with
rhBMP-2 could have clinical relevance in the future for
reducing the dose of BMP needed for new bone formation
(Toyoda et al. 2005). Similarly, in a very recent study,
Kamolratanakul et al. (2011) showed that the combined de-
livery of ONO-AE1-437 (EP4 receptor agonist) and low-dose
BMP-2, via a nanogel-based hydrogel, efficiently activated
bone cells to regenerate calvarial bone and could therefore
provide a new system for bone repair.
Ectopic bone formation was fundamental to the discovery of
BMPs in 1965 (Urist 1965). After successful production of
recombinant BMPs, it was shown that injection of recombi-
nant human BMP-2 alone into muscle tissue induced ectopic
bone formation (Wang et al. 1990). This was the basis for
further experimental investigations that led to the current
clinical utilization of recombinant BMP-2 and BMP-7 to en-
hance bone regeneration and formation.
PGE
2
and BMP-2 in osteoarthritis and cartilage
metabolism
The role of the inflammatory mediator PGE
2
in osteoarthritis
(OA) was recently investigated. Some studies have shown a
correlation between the extent of cartilage damage and the pro-
duction of IL-1␤, which induces the expression of COX-2 fol-
lowed by elevated PGE
2
levels in OA joints (Shimpo et al. 2009).
The effect of PGE
2
on chondrocytes depends on the pre
-
dominant type of EP receptor. Thus, because of the different
patterns of EP receptor expression in chondrocytes, different
effects for PGE
2
stimulation have been reported in the litera
-
ture (Aoyama et al. 2005). For example, some authors have
speculated that PGE
2
had a protective effect on articular
cartilage. Aoyama et al. (2005) found significant amounts of
EP2 receptor and minor amounts of EP3 receptor, and saw no
significant expression of EP1 and EP4 in the normal chondro-
Haversath et al. 1439
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cytes of human and mouse articular cartilage. Furthermore,
Aoyama et al. (2005) demonstrated that PGE
2
signal through
EP2 promoted the growth of articular cartilage cells in an
animal model with chondral defects. Stimulation of these
chondrocytes led to the suppression of osteopontin (OPN),
which, when increased in chondrocytes in osteoarthrotic
joints, has been linked to cartilage destruction (Yumoto et al.
2002). Nishitani et al. (2010) reported that PGE
2
inhibits
IL-1-induced extracellular matrix metalloproteinase (i.e.,
MMP-1 and MMP-13) production via EP4, suggesting a po-
tentially beneficial effect from PGE
2
on articular chondrocytes
in OA. However, numerous studies have identified catabolic,
degrading effects of PGE
2
on articular cartilage (Attur et al.
2008; Li et al. 2009). Attur et al. found the expression of all 4
EP receptor subtypes in both normal and OA chondrocytes,
with a predominance of EP4 receptors in OA chondrocytes.
Overall, they discovered catabolic effects of PGE
2
in OA
cartilage that may have been mediated via EP4 signaling
leading to increased expression of extracellular matrix degrad-
ing enzymes, such as metalloproteinases (MMP) and a disin-
tegrin and metalloproteinase with thrombospondin motifs
(ADAMTS) (Attur et al. 2008). The catabolic effect of PGE
2
in OA cartilage was also supported by Li et al. (2009), who
suggested that PGE
2
does not modulate the expression of
cartilage-degrading enzymes, but exerts its effects primarily
by inhibiting aggrecan biosynthesis in human chondrocytes
via activation of EP2. PGE
2
production, induced by mechan
-
ical shear stress on OA cartilage via COX-2, led to a signifi-
cantly increased production of IL-6, a pro-inflammatory
cytokine that is suspected to participate in the catabolic pro-
cesses associated OA (Goekoop et al. 2010).
BMPs can have opposing effects on cartilage from devel-
opment and protection, to degradation, and they are crucial for
the homeostasis of cartilage tissue. In healthy articular carti-
lage, BMP-2 can hardly be detected, whereas in OA cartilage,
BMP-2 is highly expressed (Blaney Davidson et al. 2007).
BMP-2 has been proposed to have a regenerative effect on
chondrocytes by promoting differentiation and enhancing the
expression of extracellular matrix such as type II collagen and
proteoglycan (Schmitt et al. 2003). However, initially, BMP-2
also stimulates the expression of matrix degrading enzymes
such as MMPs and ADAMTS (Blaney Davidson et al. 2007).
Blaney Davidson et al. (2007) interpreted this expression as a
short-term impulse to create space for newly produced extra-
cellular matrix. Overall, the anabolic effects of BMP-2 in
cartilage could be observed as matrix production exceeds
degradation. However, one must consider that BMP activity
alone is not sufficient to adequately protect cartilage against
destruction. Several studies have indicated that the balance of
TGF-␤/BMP signaling is of particular importance for cartilage
maintenance (Li et al. 2006). Loss of TGF-␤ signaling pro-
motes enhanced BMP signaling, which leads to terminal dif-
ferentiation of chondrocytes (hypertrophy) and results in
development of osteoarthritis (Li et al. 2006). Li et al. dem-
onstrated that BMP-2 induces maturation of chondrocytes via
R-SMAD signaling molecules (i.e., SMAD1, SMAD5, SMAD8),
whereas TGF-␤ signaling via SMAD3 inhibits maturation
and downregulates expression of BMP-2 (Li et al. 2006).
Couchourel et al. (2009) investigated normal human and OA
osteoblasts from tibial plateaus to analyze the mechanisms that
lead to undermineralized bone tissue in OA. Elevated levels of
TGF␤1 were found in OA osteoblasts. TGF␤1 is known to
have an inhibitory effect on bone formation and it exerts its
effects, at least in part, through suppression of BMP-2 pro-
duction in OA osteoblasts (Li et al. 2006). TGF␤1 also induces
enhanced, abnormal type I collagen expression in OA osteo-
blasts (Couchourel et al. 2009). Thus, undermineralized bone
tissue in OA osteoblasts may be the result of the suppression
of BMP-2 production as well as abnormal osteoid production
mediated by TGF␤1. The dual role of BMPs in articular
cartilage formation and repair and in OA development and
progression was recently described in a published review (van
der Kraan et al. 2010). BMP-2 would induce cartilage forma-
tion and repair with the expression of extracellular matrix
which can ultimately progress to chondrocyte hypertrophy,
production of matrix degrading enzymes (i.e., MMP-13) and
development of OA (van der Kraan et al. 2010).
The intertwining pathways: intracellular signaling of
PGE
2
and BMP-2 in bone
The pathways of PGE
2
and BMP-2 are closely related and
influence each other on different intra- and extra-cellular lev-
els. Understanding these intertwining pathways may be rele-
vant for the development of new therapeutic substances to
enhance bone formation and (or) cartilage metabolism. It was
shown that selective EP2 and EP4 agonists activate both the
PKA pathway and MAPK pathways (predominantly p38
MAPK and ERK) in rat calvaria cell cultures. BMP-2 effects
are mediated via R-SMADs and in part via PKA and MAPK
pathways, and this is where the signaling pathways of both
PGE
2
and BMP-2 are intertwined. Consequently, application
of EP2 and EP4 selective agonists together with BMP-2
showed an additive effect on mineralized bone nodule formation
(Minamizaki et al. 2009). Promising results for future clinical
application were recently published by Kamolratanakul et al.
(2011) who, as previously described, used nanogel-based scaf-
folds with a selective EP4 agonist (ONO-AE1-437) in com-
bination with a low dose of BMP-2 to heal critical-size bone
defects in a murine calvaria model.
Intertwining pathways in osteoblasts
The main intracellular signaling cascades in osteoblasts are
displayed in Fig. 1. Overall, both PGE
2
and BMP-2, have
cooperative anabolic effects on bone metabolism. Inflamma-
tion, along with injury to bone or soft tissue, leads to increased
production of local PGE
2
and release of BMP-2 from bone
matrix. Hypoxic conditions during injury or inflammation
stimulate PGE
2
and BMP-2 production (Tseng et al. 2010).
PGE
2
exerts its anabolic effects mainly via osteoblastic EP2
and EP4 receptor activation and increase in cAMP-levels with
activation of protein kinase A (Graham et al. 2009). The
activation results in the induction of BMP-2 and COX-2.
There is growing evidence that PGE
2
-mediated BMP-2 pro
-
duction is mainly stimulated via the EP4 receptor, while
COX-2 production is stimulated via the EP2 receptor (Sakuma
et al. 2004; Graham et al. 2009). In turn, BMP-2 induces
COX-2 expression, which leads to increased PGE
2
production
(Chikazu et al. 2002).
To summarize, the production of PGE
2
and BMP-2 in osteo
-
blasts is reciprocally promoted. Additionally, PGE
2
induces its
own production not only via EP2 and EP4 receptors, but also EP1
through an autogenous stimulation mechanism (Suda et al. 1998;
1440 Can. J. Physiol. Pharmacol. Vol. 90, 2012
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Sakuma et al. 2004). Furthermore, cAMP-signaling induces
the activation of Runx2, which is known to be an important
transcription factor promoting osteoblastic differentiation
(Yoshida et al. 2002). Recent research has suggested that EP2
and EP4 enhance Runx2 expression through cAMP-
dependent MAPK-pathways: EP2 mainly via p38 MAPK,
EP4 via ERK, both possibly promoted by protein kinase C
(PKC) (Minamizaki et al. 2009). JNK, as the third MAPK-
pathway, seems equally activated by both EP2 and EP4, which
is also involved in Runx2 activation. Furthermore, BMP-2
mediated SMAD (SMAD 1/5/8) and MAPK-signaling (p38)
are also pathways linked to the activation of Runx2 in osteo-
blasts (Ryoo et al. 2006). Additionally, SMAD signaling path-
ways also activate other transcriptional factors like Dlx5 and
Osx in both a direct and indirect manner (Ryoo et al. 2006).
Although the intertwining relations of different transcription
factors are not fully understood yet, it is recognized that the
main effect of cAMP-signaling via PGE
2
, as well as SMAD
signaling via BMP-2, is the induction of osteoblastic differen-
tiation as indicated by elevated levels of osteoblastic marker
proteins such as OPN, alkaline phosphatase (ALP), or osteo-
calcin (OCN).
The use of a selective PGE
2
EP4 agonist (ONO-4819) in
combination with BMP-2 showed a significant augmentation
of bone mass in different in vivo and in vitro studies
(Nakagawa et al. 2007). Since selective PGE
2
EP4 agonists
were found to have less of an adverse effect in animal models
compared with PGE
2
alone (Graham et al. 2009), together
with rhBMP-2 they may prove to be promising therapeutic
agents for the future enhancement of bone formation and bone
healing in humans.
Intertwining pathways in osteoblast and osteoclast
interactions
Bone formation is always coupled with the bone resorbing
process. Although the main effect of PGE
2
and BMP-2 on
bone is anabolic, osteoclastic activity can also be stimulated by
these mediators in a direct and, even more importantly, indi-
rect manner (Fig. 3). PGE
2
administration and elevated cAMP
levels have been shown to induce the expression of RANKL
and inhibition of osteoprotegerin (OPG) in osteoblasts (Jurado
et al. 2010). BMP-2 signaling via R-SMADs also stimulates
RANKL production in osteoblasts, but this effect is only
observed in the presence of PGE
2
(Blackwell et al. 2009).
However, BMP-2 alone can induce the production of colony
stimulating factor-1 (CSF-1) in osteoblasts, which is known to
promote osteoclastogenesis by activating the cFMS receptor of
osteoclasts (Mandal et al. 2009). One must consider that it is
not only PGE
2
and BMP-2 that are involved in the regulation
of RANKL-production by osteoblasts, but also other media-
tors such as parathormone (PTH), glucocorticoids, and others
(Fig. 3). PGE
2
, EP2, and EP4 receptors, as well as BMP-2,
Fig. 3. Intertwining pathways in osteoblast and osteoclast interactions. In the presence of PGE
2
, BMP-2 activates osteoblasts to produce
osteoclast-stimulating proteins such as Receptor Activator of NF-␬B Ligand (RANKL). BMP-2 also promotes osteoclastogenesis directly
via SMAD signaling and possibly via MAPK signaling, but only in the presence of RANKL. On the other hand, PGE
2
can directly inhibit
osteoclastogenesis in the absence of osteoblasts via activation of protein kinase A. During osteoclastogenesis in an inflammatory
environment, EP2 and EP4 receptors are down-regulated to maintain bone resorption.
Haversath et al. 1441
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BMPR-I, and BMPR-II receptors, were detected in osteoclasts
(Kaneko et al. 2000). Direct activation of EP2 and EP4 recep-
tors in osteoclasts has been shown to inhibit osteoclastogenesis
in the absence of osteoblasts (Mano et al. 2000). This effect
can be seen as a protective mechanism to avoid excessive bone
resorption during inflammatory conditions in the absence of
osteoblasts. On the other hand, RANKL-mediated osteoclas-
togenesis leads to down-regulation of EP2 and EP4 receptors
in osteoclasts (Kobayashi et al. 2005). This mechanism pro-
vides the maintenance of RANKL-induced osteoclastogen-
esis by preventing direct inhibition through PGE
2
. RANKL-
stimulated differentiation of osteoclasts can further be augmented
by BMP-2-mediated SMAD signaling and possibly MAPK-
signaling (Itoh et al. 2001; Jensen et al. 2010). However, without
RANKL, BMP-2 does not induce osteoclastogenesis.
To summarize, PGE
2
can induce the expression of RANKL
in osteoblasts, leading to osteoclastic differentiation further
enhanced by the co-presence of RANKL and BMP-2. On the
other hand, PGE
2
can also directly inhibit osteoclastogenesis
through the activation of EP2 and EP4 receptors on oste-
oclasts. However, this inhibition is counter-balanced by the
down-regulation of these receptors by RANKL-mediated os-
teoclastogenesis.
In the absence of PGE
2
and RANKL, BMP-2-induced stim
-
ulation of osteoclasts can be mediated indirectly via CSF-1
production in osteoblasts. However, BMP-2 alone does not
affect the production of RANKL in osteoblasts (Blackwell et
al. 2009).
Conclusions
Inflammation processes and bone remodelling are strongly
interconnected. The overall effect of PGE
2
and BMP-2 on
bone is anabolic. Recent research has demonstrated that the
intracellular signaling pathways of both mediators are inter-
twined and that their production in osteoblasts is reciprocally
promoted. Contrary to PGE
2
, selective PGE
2
EP4 receptor
agonists caused no severe side effects while providing similar
anabolic effects. Hence, EP4 agonists together with rhBMPs,
and in particular rhBMP-2, may be promising therapeutic
agents for enhancing bone formation and bone healing in
human skeletal disorders.
Competing interests
The authors declare that there is no conflict of interest
associated with this work.
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