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Dexamethasone Use During Pregnancy: Potential

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Current Pharmaceutical Design, 2014, 20, 000-000 1

Dexamethasone Use During Pregnancy: Potential Adverse Effects on Embryonic

Skeletogenesis

Xin Cheng1*, Guang Wang1, Kenneth Ka Ho Lee2 and Xuesong Yang1,3*

1
Department of Histology & Embryology, Joint Lab for Brain Function and Health, School of Medicine, Jinan University, Guangzhou
510632, China; 2Key Laboratory for Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong,
Shatin, Hong Kong; 3Institute of Fetal-Preterm Labor Medicine, Jinan University, Guangzhou 510632, China

Abstract: Glucocorticoids are important regulators of cell differentiation and mesenchymal cell lineage commitment during skeletogene-
sis. In clinical practice, it has been difficult to study the effects of glucocorticoids on target tissues because patients taking glucocorticoids
often suffer from adverse skeletal effects. Dexamethasone (Dex) is a long-acting synthetic corticosteroid hormone that ranks amongst the
most widely used prescribed drugs, and it is a powerful medication that is increasingly employed during the perinatal and neonatal peri-
ods. However, Dex is a potential teratogen. In particular, it has been claimed that Dex exposure during pregnancy can affect osteogenesis
in the developing embryo, although this claim remains highly controversial. In this review, we summarize the published data from nu-
merous clinical follow-up, animal-based and in vitro studies on the effects of Dex exposure on embryonic skeletogenesis. These studies
indicate that Dex may adversely affect skeletal progenitor cells during development. In addition, Dex can exert a number of effects on
bone growth at different developmental stages. We also discuss how glucocorticoids influence the BMP, FGF, Hedgehog and Wnt signal-
ing pathways, which are key regulators of skeletogenesis in the embryo. A fuller understanding of the negative, and perhaps teratogenic,
effects of Dex on skeletogenesis will have important implications for the routine use of Dex in clinical practice.
Keywords: Glucocorticoids, dexamethasone (Dex), chondrogenesis, osteogenesis, signal transduction pathways.

INTRODUCTION
Glucocorticoids are considered important regulators of os-
teogenic cell differentiation and mesenchymal cell lineage com-
mitment. However, the functional effects of glucocorticoids on
target tissues and organs are still not fully understood, and patients
taking glucocorticoids often suffer from harmful side effects. For
example, children that are given glucocorticoids show reduced bone
quality (also observed in adults), which can impact their growth and
stature [1]. It has been reported that the treatment of childhood
asthma, autoimmune diseases and pediatric cancers with the long-
term administration of anti-inflammatory glucocorticoids can result
in growth retardation, bone loss and potentially premature or severe
osteoporosis. In addition, there is a growing body of evidence
showing that even short-term administration of glucocorticoids can
alter bone growth and turnover and that these effects can vary de-
pending on how frequently (e.g., single or repeated dose) glucocor-
ticoids are administered [2-7]. On the other hand, a fetus is nor-
mally exposed to elevated levels of endogenous glucocorticoids
during the late gestational period, which is essential for the adaption
and maturation of developing bone during postnatal life.
Dexamethasone (Dex), a synthetic long-acting corticosteroid
hormone, is one of the most widely prescribed drugs for the treat-
ment of inflammatory disorders such as arthritis, swelling, redness
of the skin, adrenal hormone insufficiency, resistance to adrenal
hormones, and asthma and kidney disorders. Furthermore, Dex
induces the expression of the xenobiotic-metabolizing enzyme CYP
in the liver, and it has therefore also been used to amplify the ef-
fects of anti-cancer drugs [8] and as an illicit enhancer of athletic
performance.
In the clinical setting, Dex is one of the most powerful drugs
prescribed during the perinatal and neonatal periods, and its use is
*Address correspondence to these authors at the Division of Histology &
Embryology, Medical College, Jinan University, No. 601 Huangpu Road
West, Guangzhou 510632, PR China; Tel: +86-20-85228316, E-mail:
yang_xuesong@126.com
Tel: +86-20-85220254, E-mail: chengxin633@gmail.com.cn
increasing. Dex has also been used to reduce the risk of neonatal
respiratory distress syndrome and as a medication for several types
of severe pregnancy disorders, including congenital adrenal hyper-
plasia, premature delivery caused by pregnancy-induced hyperten-
sion (PIH), placenta previa and multiple pregnancies [6, 9-12].
However, certain data suggest that human fetal exposure to Dex has
detrimental effects on birth outcome, childhood cognition and long-
term behavior, the ramifications of which may be permanent. In
addition, the prenatal administration of Dex can affect lifespan and
predisposition to chronic diseases [13]. Therefore, opinions differ
on whether glucocorticoids should be used therapeutically in pre-
mature infants to prevent prematurity-related complications and to
treat neonatal respiratory distress syndrome. Despite this, Dex re-
mains widely used in primary hospitals as one of the most effective,
long-term glucocorticoids.
As the use of Dex has been extensive, it is extremely important
that we understand any potentially harmful side effects it may have
on embryonic development. The effects of Dex on adults and ado-
lescents have been much better studied compared with its effects on
embryos, and we know relatively little of how Dex exposure affects
signaling molecules and pathways during development. Moreover,
the majority of current studies address the neurotoxic effects of Dex
during development, rather than discussing the merits and demerits
of Dex treatment during the perinatal period [7, 14-19]. In addition,
there are relatively few reports on the consequences of Dex expo-
sure on fetal bone growth. Therefore, the aim of this review is to
provide information on the effects of Dex usage on skeletogenesis
during the prenatal period and to highlight previously published
clinical follow-up, animal-based and in vitro studies.
VERTEBRATE SKELETOGENESIS
Bones are normally formed through either intramembranous
ossification or endochondral ossification, which are distinct but
related morphogenetic processes. During intramembranous ossifica-
tion – which is how the bones in the skull, jaw and collarbones are
formed – richly vascularized mesenchymal cells condense to form
nodules and flat plates that then directly differentiate into os-
teoblasts. However, the vertebrate skeleton primarily develops via

1381-6128/14 $58.00+.00 © 2014 Bentham Science Publishers

2 Current Pharmaceutical Design, 2014, Vol. 20, No. 00
endochondral ossification, which is a well-orchestrated and intri-
cately controlled set of processes that includes the condensation,
proliferation, migration, differentiation and activation of multiple
cell types [20-22]. In particular, endochondral ossification is how
long bones and the growth plates within long bones develop during
the embryonic and postnatal periods, respectively. In addition, os-
teogenesis occurs not only in growing bones but also throughout
adult life, and it is involved in the formation of primary and secon-
dary ossification centers as well as in bone absorption. Indeed, bone
turnover is very active in young children and can occur up to 200
times as quickly as it does in adults. Nevertheless, the most crucial
and active period of osteogenesis is during embryogenesis.
THE EFFECTS OF DEXAMETHASONE ON PROGENITOR
CELLS DURING SKELETAL DEVELOPMENT IN THE
EMBRYO
The vertebrate skeleton is derived from three cell populations
during embryonic development. Neural crest cells give rise to the
craniofacial bones [23], the sclerotome compartments of somites
form the axial skeleton [24], and lateral plate mesoderm cells form
the limb bones [25].
NEURAL CREST CELLS
During the early stages of embryonic development, neural crest
cell derivatives become responsive to glucocorticoids once the
sympathetic ganglia are formed, and Dex plays a crucial role in the
migration and differentiation of these neural crest cells. Several
retrospective studies have been carried out on infants born to
women who had received multiple courses of glucocorticoids dur-
ing pregnancy, and these studies reported reduced body weight and
head circumference at birth in these children. Furthermore, a 3-year
follow-up study showed similar findings for children exposed to
only a single course of glucocorticoids during development [6].
Indeed, a single course of Dex is considered sufficient for prevent-
ing prematurity-related complications. It has been reported that
exposure to Dex during early gestational stages can cause minor
cranial-skeletal abnormalities, including encephalocele and menin-
gocele, which was also observed in rhesus macaques [26]. These
findings suggest that the development and migration of neural crest
cells may be affected by Dex exposure during early gestation. Neu-
ral crest cells also contribute to the formation of the palate, and
animal models have been developed that use hydrocortisone, pred-
nisolone and Dex on pregnant to produce embryos with cleft pal-
ates. From these studies, it was shown that the teratogenicity of Dex
is 300 times greater than that of hydrocortisone for inducing cleft
palate [27]. For this reason, Dex has been widely employed in ani-
mal models for inducing cleft palate in vivo, although the precise
mechanism through which Dex induces this abnormality is not
clear. It is known that the adhesion and fusion of the palatal shelves
are necessary for the proper development of the secondary palate,
and failures in these processes can lead to cleft palate. In the fetuses
of mothers exposed to Dex, the elevation of the palatal shelves is
delayed and the descent of the tongue is interrupted, which impedes
the rotation of the shelves, leading to a failure in the fusion of the
shelves. Furthermore, the cell death and proliferation that normally
occur at the medial edge of the palatal epithelium can also be inhib-
ited by Dex exposure during development, leading to altered cell
fates, small palate size, delayed shelf elevation and unfused palates,
which may be the most important mechanism underlying the failure
of palatal shelf fusion [28].
As Dex is a glucocorticoid receptor (GR) agonist, its effects on
embryonic development can be elucidated by studying the GR. The
spatial-temporal expression pattern of GR in the early Xenopus
embryo has been systematically mapped [29], and it was found that
GR mRNAs are localized to the dorsal ectoderm during the gastrula
stage and to the notoplate during the early neurula stage; GR ex-
pression is lost sometime between the middle and late neurula
Cheng et al.
stages. At the tailbud stage, GR is expressed in the anterior portion
of the embryo, including the somites. Furthermore, when zygotes
over-expressing GR were treated with Dex at the blastula stage,
early differentiation was significantly inhibited.
EMBRYONIC SOMITES
Hansen et al. (1994) examined the toxicity of Dex in mouse and
rat embryos, and they observed a significant reduction in the num-
ber of somite pairs produced when embryos were treated with Dex.
As somites differentiate to form the sclerotome, which will eventu-
ally form the vertebrae, it was therefore not surprising that Dex
treatment led to malformations of the axial skeleton. The crown-
rump and head lengths of these embryos were also significantly
reduced. In addition, these researchers reported that mouse embryos
cultured in vitro were more adversely affected by Dex than were rat
embryos, even at lower concentrations [30]. This difference in the
sensitivity of these two species to Dex may be attributed to inherent
genetic differences or to minor differences in the developmental
staging of the embryos used in the culturing experiments.
Glucocorticoids exert their effects primarily through the GR,
and these receptors are extensively expressed in mammalian tissues,
including bone growth plates [31, 32]. Many studies have been
conducted to examine how glucocorticoids regulate skeletogenesis,
and these studies indicate that glucocorticoids affect the formation
of hyaline cartilage, which forms the prerequisite limb scaffolding
necessary for endochondral ossification in the long bones. Studies
involving the use of a GR morpholino on developing embryos
showed that it had the ability to delay somitogenesis, induce somite
and tail malformations, and reduce embryo size. A key finding of
this study was the 70-90% reduction in bone morphogenetic pro-
teins (BMPs) expression observed following GR-morpholino treat-
ment. Further molecular analysis identified multiple putative gluco-
corticoid response elements upstream of the BMP genes, and it was
shown that Exposure to the GR morpholino reduced expression of
the BMP-regulated genes eve1 and pax3 [33].
The release of cells and tissues from growth-inhibitory condi-
tions generally results in compensatory growth in mammals. In
piglets, it has been shown that prenatal exposure to Dex during the
last 24 days of the fetal period resulted in a dramatic reduction in
bone mineral density and content. In addition, they observed de-
creases in blood serum osteocalcin levels as well as in various bone
geometric parameters, ultimately increasing the risk of these piglets
for developing bone fractures [34, 35]. Although prenatal Dex
treatment did not reduce birth weight or growth rate during the first
30 days post-birth, no compensatory catch-up growth in terms of
bone mineralization was observed in these suckling piglets [34].
EMBRYONIC LIMB BUDS
The limb develops as an outgrowth, or limb bud, from the flank
of the embryo consisting of the lateral plate mesoderm. The limb
bud is composed of mesenchymal cells covered by a single layer of
ectoderm. At the distal end of the bud, the ectoderm thickens and
forms a multilayered cellular structure called the apical ectodermal
ridge (AER) [36]. The AER secretes signaling molecules that
stimulate the underlying mesenchymal cells to rapidly proliferate
while maintaining a multipotent state. This mesenchymal region is
known as the progress zone, and these cells eventually differentiate
into cartilage, perichondrium, tendon, muscle connective tissue and
dermis. The AER directs the mesenchyme in the progress zone to
differentiate into chondrogenic and non-chondrogenic precursors.
The limb bud forms a chondrogenic core and is surrounded periph-
erally by soft connective tissue. Limb myogenic cells, which origi-
nate from the somites, migrate into the limb bud and differentiate
into skeletal muscles in a pattern that is directed by the connective
tissues. Highly coordinated signaling between the AER and the
underlying mesenchymal cells specifies the distal structures of the
limb and also maintains the survival of the mesenchymal cells. In
Dexamethasone Use During Pregnancy
particular, the signaling genes involved in this process are Shh,
Hox, Fgf10, Fgf8, Tbx4, Tbx5 and the BMPs, which must be ex-
pressed in a precise spatiotemporal fashion for the limbs to develop
and pattern properly [37-40]. However, there are few reports in the
literature of the effects of glucocorticoids on limb bud develop-
ment.
THE REGULATORY EFFECTS OF DEXAMETHASONE ON
DIFFERENT STAGES OF BONE DEVELOPMENT
Endochondral ossification is composed of two crucial morpho-
genetic stages. First, the mesenchymal progenitor cells condense
into a nodule, which is then followed by the differentiation of these
cells into chondroblasts and perichondrium in a process called
chondrogenesis. Next, the cartilage scaffolding is replaced by mi-
crovasculature, osteoblasts, osteoclasts, osteocytes and bone matrix,
ultimately giving rise to bone. Crucially, bone growth depends on
the tight coordination between these two morphogenetic processes.
Glucocorticoids have been implicated in the regulation of chondro-
genesis and osteoblast differentiation, as well as in maintaining the
homeostasis of cartilage and bone at a physiological level.
CHONDROGENESIS
Pharmacological doses of glucocorticoids have been shown to
inhibit chondrocyte proliferation within the growth plate, and this is
one of the major side effects of glucocorticoids that suppress longi-
tudinal bone growth. More specifically, Dex exposure significantly
reduces cell number, cell proliferation, and proteoglycan synthesis
and increases alkaline phosphatase (ALP) activity during the chon-
drogenesis in the ATDC5 cell line, which mimics the in vivo proc-
ess of longitudinal bone growth; apoptosis was unaltered in this
model [41]. On the other hand, Dex has been used in combination
with transforming growth factor beta-1 (TGF-
1) to induce the
differentiation of mesenchymal stem cells (MSCs) from a variety of
tissues into chondrocytes [42-44]. In fact, the effects of Dex on the
chondrogenic differentiation of MSCs is influenced by microenvi-
ronment and tissue source, as well as by the nature of the particular
growth factor used [45], suggesting that Dex is necessary for MSC
chondrogenesis. Some of the regulatory targets of glucocorticoids
during chondrogenesis are now known [46], and they include ex-
tracellular matrix (ECM)-related, metabolic, cytokine and growth-
factor genes. However, the global effects of glucocorticoids (at
pharmacological doses) on chondrocyte gene expression have not
yet been comprehensively evaluated.
OSSIFICATION
During endochondral bone formation, terminal differentiation
of chondrocytes involves a number of steps: the rate of cell prolif-
eration decreases; the chondrocytes become hypertrophic; the ECM
surrounding the hypertrophic chondrocytes becomes progressively
mineralized; and finally, the mineralized matrix serves as a tem-
plate for bone deposition. This chondrocyte maturation process is
precisely controlled, with chondrocyte proliferation and terminal
differentiation being tightly balanced, and indeed, imbalances can
result in an abnormal bone development. In general, the degree of
chondrocyte maturation is evaluated by measuring the levels of
ALP expression and the presence of type X collagen within the
mineralized matrix.
Leclerc et al. (2004) revealed that Dex could arrest osteoblast
development in MC3T3-E1 cell cultures [47]. In contrast, Dex had
little effect on terminal differentiation in the chondrocyte cell line
ATDC-5 [41]. It appears that the ability of Dex to inhibit osteoblast
development is strongly dependent on concentration and the timing
of administration, which suggests that there is a specific period of
skeletogenesis during which that tissue is sensitive to Dex expo-
sure.
A comparative microarray analysis was conducted on mouse
embryonic chondrocytes treated with a pharmacological dose (10-7
Current Pharmaceutical Design, 2014, Vol. 20, No. 00 3
M) of Dex. It appears that Dex acts on cartilage by regulating mul-
tiple classes of genes to promote the accumulation of ECM and the
activation of metabolic-related genes to maintain the chondrocytic
phenotype. At the same time, Dex repressed cytokine and growth
factor synthesis, which can accelerate the shift from cartilage to
bone phenotypes. In addition, Dex-induced gene expression data
have been compared with gene expression data from various devel-
opmental phases of micromass cultures. These results indicate that
Dex maintained the expression of various chondrocytic marker
genes while inhibiting factors that promote the vascularization and
ossification of the cartilaginous anlagen [48]. These studies suggest
that Dex exerts its effects in a tissue-specific manner, and determin-
ing the precise network of genes influenced by Dex treatment
should allow us to understand the mechanisms through which Dex
affects vertebrate skeletogenesis.
THE INFLUENCE OF DEXAMETHASONE ON SIGNAL
TRANSDUCTION AND SKELETOGENESIS
A large number of studies have implicated several signaling
molecules, including growth factors and hormones, in the regula-
tion of skeletogenesis. Chondrocytes and osteoblasts constantly
receive signals from surrounding cells and tissues, and even from
distant organs, that regulate their proliferation, biological activity
and survival. Two of the fundamental questions of interest to devel-
opmental biologists are how undifferentiated precursor cells acquire
spatial patterning information, and in turn, how these cells differen-
tiate and respond to the information provided. The developing ver-
tebrate limb has been extensively studied as a model for addressing
these questions, and as a consequence, the body of knowledge gen-
erated by such studies has provided novel insights into the mecha-
nisms governing the development of skeletal phenotypes and the
signaling pathways involved. Nevertheless, the global regulatory
effects of glucocorticoids on the expression of key skeletal-related
genes has not been comprehensively evaluated, and in particular,
how these signaling pathways are spatiotemporally regulated to
orchestrate growth and differentiation during embryogenesis re-
mains unclear. Ultimately, we wish to know how the disruption of
these signaling pathways, through either genetic mutations and/or
environmental factors, leads to skeletal malformations. The impor-
tance of the precise regulation of signal transduction for normal
development is highlighted by the observation that these pathways
are often misregulated in adult diseases and congenital malforma-
tions. These findings are summarized here, and the important fea-
tures of the primary signaling pathways will be discussed as they
relate to glucocorticoids and Dex-mediated skeletogenesis.
BONE MORPHOGENETIC PROTEINS
The BMPs are a family of secreted proteins that have multi-
functional roles in specifying the dorsal-ventral axis embryos and in
determining cell fate at the neural plate and during limb bud devel-
opment [49]. BMPs induce a variety of biological activities in an
assortment of cell types at various stages of differentiation, al-
though their roles during normal physiology are still not completely
clear. Nevertheless, there is no doubt that BMPs play an essential
role in endochondral bone development [50].
When mesenchymal progenitor cells are treated with Dex, it
induces these cells to differentiate into chondroblasts or osteoblasts.
However, the effect of Dex on cell differentiation is enhanced in the
presence of BMP-2 [51, 52]. In addition, the decision made by pro-
genitor cells to differentiate into either chondroblasts or osteoblasts
is dependent on the microenvironment and whether other inducers
are also present [53-55]. It has also been reported that BMP-7 is
expressed by proliferating chondrocytes [56].
As described above, chondrocyte maturation is precisely con-
trolled to yield a balance between proliferation and terminal differ-
entiation during endochondral ossification. BMPs exert profound
effects on chondrocyte maturation, and BMP-2 and BMP-6 are
4 Current Pharmaceutical Design, 2014, Vol. 20, No. 00
expressed in hypertrophic chondrocytes. Microarray analysis of
Dex-arrested osteoblasts in MC3T3-E1 cells revealed that BMP
biological activity was inhibited as a result of the strong repression
of Krox20/Egr2. Furthermore, when Follistatin (a BMP antagonist)
was over-expressed in cells, it negatively regulated by Krox20 [47].
When ascorbate and BMP-2 inducers are combined and added to
sternal pre-hypertrophic chondrocytes, they induced a threefold
increase in ALP levels compared with controls [57]. Minina 2001
reported that the addition of BMP-2 to mouse and chick limb ex-
plant cultures dramatically increased chondrocyte proliferation and
delayed terminal differentiation (i.e., chondrocyte hypertrophy)
[58]. It is possible that these different observed effects for inducers
containing the same signaling molecules is partially due to the dif-
ferent drug concentrations used or the specific culturing systems
and manipulation protocols.
INDIAN HEDGEHOG AND PARATHYROID HORMONE-
RELATED PROTEINS
During endochondral ossification, the Indian hedgehog (Ihh)
and parathyroid hormone-related (PTHrP) proteins have been iden-
tified as components of the negative feedback loop that regulates
chondrocyte hypertrophic differentiation. Ihh is a dominant regula-
tor of bone development and is involved in coordinating chondro-
cyte proliferation and differentiation, as well as osteoblast differen-
tiation. This protein directly stimulates chondrocyte proliferation
and determines the length of the proliferating chondrocyte columns
through the stimulation of PTHrP synthesis. Furthermore, Ihh ex-
pression also defines the sites where hypertrophic differentiation
begins. In the cartilage growth plate, the biological activities of
mature chondrocytes and early hypertrophic chondrocytes are pre-
cisely regulated by Ihh and PTHrP, which is essential for normal
longitudinal growth.
Ihh is closely related to Sonic hedgehog (Shh), one of the main
regulators of limb bud outgrowth. During endochondral bone de-
velopment, Ihh is synthesized by early hypertrophic chondrocytes
and by chondrocytes just leaving the proliferative zone, which are
known as prehypertrophic chondrocytes. When MSCs are exposed
to low doses of Dex (10-8 M), they differentiate into osteoblasts,
which is indicated by enhanced ALP activity and the production of
collagen type I. Dex can also enhance Shh expression via a Gli1-
independent mechanism during osteoblast differentiation in MSCs.
The expression of both Ihh and Gli1 is down-regulated during this
process [59]. These findings partially support the observations of
Heine and Rowitch [14] that chronic treatment of mouse pups with
glucocorticoids inhibits neonatal cerebellar growth and neuronal
proliferation by promoting early cell cycle exit and premature dif-
ferentiation. In vitro analysis revealed that glucocorticoids re-
pressed Gli1 (a target of Shh) expression, suggesting the existence
of a mutual antagonism between glucocorticoids and Shh and that
different patterns of hedgehog signaling are involved in Dex-
induced development.
Studies involving transgenic mice that specifically over-
expressed Ihh in chondrocytes revealed that PTHrP expression was
up-regulated in these cells and that there was a delay in the onset of
hypertrophic differentiation. In general, it is thought that BMPs are
potential integrators of the Ihh/PTHrP feedback loop. Treatment of
the limbs obtained from these transgenic mice with Noggin (an
inhibitor of both BMP and Ihh/PTHrP signaling) revealed that this
molecule did not antagonize the effects of Ihh overexpression.
Conversely, the enhancement of chondrocyte maturation induced
by cyclopamine (an inhibitor of Ihh signaling) could not be re-
versed by BMP-2. These observations suggest that BMP signaling
alone delays hypertrophic differentiation, independent of the
Ihh/PTHrP signaling pathway. Or in other words, BMP signaling
does not act as a downstream signal of Ihh/PTHrP to delay the on-
set of hypertrophic differentiation [58].
Cheng et al.
FIBROBLAST GROWTH FACTORS
The fibroblast growth factor (FGF) family is composed of 22
distinct FGF genes and 4 FGF receptor genes. These molecules are
expressed at nearly every stage of bone formation, and they un-
doubtedly play key roles in the regulation of bone development.
During the earliest stages of bone development, FGF receptor-1
(FGFR1) is expressed in prehypertrophic/hypertrophic chondro-
cytes and in the perichondrium. FGFR2 is expressed in the chon-
drogenic condensation, perichondrium, periosteum and primary
spongiosa. FGFR3 is expressed in proliferating chondrocytes. Fi-
nally, FGF-7, -8 -17 and -18 are expressed in the perichondrium
[60]. Each of these growth factors appears to play distinct and im-
portant roles in bone development, although their exact roles have
not been fully defined. FGF-2, platelet-derived growth factors
(PDGFs) and TGF-
1 have long been recognized as inducers of
chondrogenic phenotypes in MSCs, and they have been used to
generate cartilage grafts [53, 61-64]. This process requires specific
culture conditions that permit rapid the rapid expansion of MSCs
while maintaining the potential of these cells for further differentia-
tion. Dex is routinely added to MSC cultures to promote cell prolif-
eration and cell pellet expansion, and these effects can be enhanced
when combined with a low concentration of FGF-2 (1 ng/mL) [65-
67]. Dex enhances chondrogenesis and osteogenesis in pellet cul-
tures by increasing nodule formation, glycosaminoglycan deposi-
tion, collagen type II synthesis and SOX9 expression. It also posi-
tively promotes cell maturation by increasing expression of the
developmental markers STRO-1 and ALP [67, 68]. MSCs ex-
panded with Dex/FGF-2 display lower ALP levels than Dex-
expanded cells but significantly higher ALP levels than FGF-2-
expanded cells [67]. These results suggest that both Dex and FGF-2
are crucial components in the maintenance of osteogenic potential,
although their exact roles are still obscure.
Among the FGF receptors, the function of FGFR3 is most fully
understood. FGF signaling is able to inhibit cell proliferation
through FGFR3. When Fgfr3 is knocked out in mice, it leads to
severe and progressive bone dysplasia with enhanced and pro-
longed endochondral bone growth. This phenotype is accompanied
by the expansion of proliferating chondrocyte columns and hyper-
trophic chondrocytes within the cartilaginous growth plate [69]. In
humans, Fgfr3 mutations cause achondroplasia, which is the most
common type of dwarfism [70, 71]. Knocking out Fgf-18 in mice
also leads to increased chondrocyte proliferation and delayed ossi-
fication, similar to what is observed in Fgfr3-knockout mice [72].
Therefore, FGF18 is thought to play a role in FGFR3 signaling.
D8-SBMC is a cell line established from rat bone marrow that
is capable of producing bone-like nodules and robust mineralization
when induced with FGF-2 and Dex. When AJ18 (a regulator of
bone formation that is up-regulated by BMP-7) is over-expressed in
D8-SBMC cells, it increases cell proliferation and mineralization.
Expression of bone-related gene markers is also up-regulated by
AJ18 over-expression [73]. Furthermore, the biphasic modulatory
effects of AJ18 on cell proliferation and mineralization are associ-
ated with BMP-7.
Each of the signaling systems mentioned above is essential,
although not sufficient, for bone formation; in other words, loss of
any of these systems can prevent bone from developing normally.
These signaling systems influence and interact with one another to
provide the developing bone with precise patterning information for
further growth. FGF signaling shortens chondrogenic proliferative
columns in the cartilage scaffolding by directly inhibiting chondro-
cyte proliferation and suppressing Ihh expression. When Fgfr3 is
knocked out, it increases Ihh expression, whereas activation of
FGFR3 decreases Ihh expression. FGF signaling can also accelerate
the terminal differentiation of hypertrophic chondrocytes independ-
ently of Ihh and PTHrP. BMPs can oppose the effects of FGF sig-
naling by increasing chondrocyte proliferation and inducing Ihh
expression. BMPs and FGFs have opposing effects on terminal
Dexamethasone Use During Pregnancy
hypertrophic chondrocyte differentiation, and these pathways can
be considered as antagonistic at several levels [74]. In this context,
chondrocyte proliferation, osteoblast differentiation and hypertro-
phic differentiation are the result of antagonistic interactions be-
tween several signaling pathways. Therefore, simply increasing or
decreasing one of these signaling systems cannot account for the
variety of phenotypes observed during skeletogenesis.
WNT PROTEIN LIGAND
In humans, the Wnt family is composed of 19 members. They
are involved cell-cell communication and are essential for normal
embryonic development and differentiation. The Wnt signaling
pathway is activated when Wnt protein ligands bind to a Frizzled
family receptor protein. These signaling pathways are activated in a
highly coordinated manner to provide positional information to
cells in order to determine their proper identity and developmental
fate and ensure proper embryonic patterning. Wnts affect many
diverse processes, including embryonic induction, generation of cell
polarity and cell fate specification [75]. During early craniofacial
development in mouse embryos, Wnt family members are ex-
pressed in a variety of locations, including the tooth initiation sites
of the oral epithelium, the mesenchyme of the elevating palatal
shelves, and the prospective sites of the maxilla and mandible
bones. The Wnt ligands function by activating both Wnt/-Catenin-
dependent and -Catenin-independent pathways. It has been re-
ported that Wnt/-Catenin signaling was down-regulated in a Dex-
induced cleft palate mouse embryo model and that the downstream
molecules of Wnt/-catenin signaling, including -catenin, Lef-1, c-
Jun and phospho-c-Jun, were completely inhibited by Dex treat-
ment. Indeed, altered signaling in the affected cells directly im-
paired their ability to form a normal palate [28]. In the limb bud,
Wnt ligands secreted by the ectoderm promote the underlying mes-
enchymal cells to proliferate via the activity of Nmyc and inhibit
chondrogenic differentiation via the repression of Sox9 [37].
Col2.3-11HSD2 transgenic mice, which are characterized by
osteoblast-targeted disruption of glucocorticoid signaling, have
been used as a model to study the relationship between skeleto-
genesis and glucocorticoids [76-78]. Micro-CT scans of Col2.3-
11HSD2 fetuses and neonates revealed they had hypoplasia and
osteopenia of the skull bones, disorganized frontal and parietal
bones, increased suture patency, ectopic cartilage in the sagittal
suture, and disrupted postnatal removal of parietal cartilage. These
transgenic mice showed markedly reduced levels of Mmp14, an
enzyme essential for calvarial cartilage removal. Wnt9a and
Wnt10b expression was also significantly reduced in osteoblasts
with disrupted glucocorticoid signaling, which in turn caused a
reduction in -catenin (an upstream regulator of Mmp14) accumu-
lation within osteoblasts, chondrocytes and mesenchymal progeni-
tors [78]. These observations suggest that intramembranous ossifi-
cation is tightly regulated by glucocorticoids and that this hormone
initiates and controls cartilage dissolution after birth. These findings
agree with experiments conducted on transgenic mice lacking Axin2
(a negative regulator of Wnts) and mutant for Fgfr1, which leads to
the development of craniosynostosis. It appears that switching mes-
enchymal cell fate from osteoblasts to chondroblasts resulted in
suture abnormalities, suggesting that ectopic endochondral ossifica-
tion may be a mechanism for producing craniosynostosis [79].
Therefore, the balance between Wnt and FGF signaling influences
the developmental fate of mesenchymal cells during skeletogenesis.
It has been reported that calvarial cell cultures produced from
Col2.3-11HSD2 mice were more prone to undergo adipogenesis
than osteoblastogenesis. This change in cell fate commitment by
Col2.3-11HSD2 progenitor cells was associated with reductions in
Wnt7b, Wnt10b and -catenin expression. However, co-culturing
these transgenic cells with wild-type osteoblasts restored the ten-
dency of these cells to form osteoblasts. This effect could be
blocked by the introduction sFRP1, a Wnt inhibitor, to the co-
Current Pharmaceutical Design, 2014, Vol. 20, No. 00 5
cultures. Moreover, treatment of the transgenic cultures with Wnt3a
was shown to stimulate osteoblastogenesis and inhibit adipogenesis
[80]. Taken together, these results demonstrate a central role for
osteoblasts in the regulation of early lineage commitment in pro-
genitor cells, which can be blocked by the loss of Wnt signaling.
Cells that express Wnt3a often show enhanced proliferation and cell
survival. Wnt3a can also up-regulate ALP expression without influ-
encing matrix mineralization but represses most of the other genes
associated with osteogenic differentiation, which can be partially
reversed by BMP-2 [81]. Furthermore, the abnormalities observed
in Col2.3-11HSD2 mice can be rescued by supracalvarial injection
of Wnt3a protein [78], demonstrating the role of Wnt3a in os-
teoblastogenetic potential. The up-regulation of ALP expression
induced by Wnt-3a could be effectively suppressed through the
combined activity of osteogenic supplements, Dex and 1,25-
dihydroxyvitamin D (1,25(OH)2D3) [78, 81].
Glucocorticoids exert both anabolic and catabolic effects on
bone, which are partly mediated by Wnt signaling molecules and
their inhibitors in mature osteoblasts [82]. The mRNA levels of
Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt10b, and Wnt11 are significantly
higher in osteoblasts than in their progenitor cells. In particular, the
expression of Wnt7b and Wnt10b in osteoblasts is modulated by
glucocorticoids in a biphasic manner, with both genes being up-
regulated at low corticosterone concentrations and down-regulated
at high concentrations. High concentrations of glucocorticoids also
increase the expression of the Wnt inhibitors sFRP-1 and DKK-1.
GROWTH HORMONE AND INSULIN-LIKE GROWTH
FACTOR-I
The growth inhibition caused by glucocorticoid therapy during
development is thought to be mediated by the somatotropic hor-
mone axis and by direct local effects on growth plate chondrocytes.
Therefore, changes in the secretion of growth hormone (GH) could
impair the longitudinal growth of the chondrocyte plate. The inter-
actions between glucocorticoids, GH and parathyroid hormone
(PTH) have recently been investigated in detail. In growing chon-
drocytes cultures, the introduction of GH, PTH and 1,25(OH)2D3
increases chondrocyte proliferation by stimulating insulin-like
growth factor-1 (IGF-1) secretion in a paracrine fashion. Long-term
high-doses of glucocorticoids decreased GH secretion, whereas
PTH and 1,25(OH)2D3 stimulated cell growth in a dose-dependent
manner [83]. Glucocorticoids at high doses reduced GH receptor
and IGF type-1 receptor (IGF-1R) expression. However, the pri-
mary anti-proliferative effect of the glucocorticoids was to reduce
the basal levels of hormone-stimulated IGF-1 secretion by human
osteoblast-like cells [84]. These in vitro results are consistent with
observations of animal models and children treated with glucocorti-
coids showing that the inhibitory effects of glucocorticoids on bone
formation in humans are mediated via a reduction in the
autocrine/paracrine expression of IGF-1, which can be compensated
for by supra-physiological levels of GH or IGF-1 [85].
Treatment with Dex, either continuously or on alternating days,
induced the same degree of growth retardation in fetal metatarsal
cultures. Dex inhibited cell proliferation within the growing bone,
whereas IGF-1, either alone or in combination with Dex, induced
an increase in linear bone growth. GH alone at 100 ng/mL exerted
no beneficial effects on metatarsal growth. However, IGF-1 signifi-
cantly increased the length of hypertrophic zone, whereas Dex
alone had no significant effect [86]. Treatment of osteogenic pre-
cursors with IGF-1, either alone or in combination with Dex, had no
significant effects on colony formation or the expression of ALP
and the developmental marker STRO-1. In contrast, exposing these
same cells to FGF-2 increased colony formation, cell proliferation
and expression of STRO-1/ALP. Normally, the
and  subunits of
IGF-1R are expressed in the majority of osteoprogenitor cells. Dex
treatment did not affect IGF-1R expression, although it did increase
the number of cells co-expressing IGF-1 and ALP [87]. Taken to-
6 Current Pharmaceutical Design, 2014, Vol. 20, No. 00 Cheng et al.

Fig. (1). Dex exerts distinct effects at every stage of embryonic skeletogenesis. The vertebrate skeleton is derived from three cell populations: neural crest
cells, somites and limb buds. Dex reduces body weight and head circumference, causes cranial skeletal abnormalities and cleft palate
, reduces somite pair
number and embryo size, delays somitogenesis, and induces somite and tail malformations
; the precise effects of Dex on limb buds remains unclear
.
Dex regulates proliferation and differentiation in chondrocytes

and the process of bone ossification , although the effects are variable and may be de-
pendent on micro-environment. Such changes in proliferation and differentiation may account for the cellular mechanisms underlying the phenotypes caused
by Dex treatment. The global effects of Dex on key skeletal-related signaling pathways have not been comprehensively evaluated. In general, FGFs act to
decrease chondrocyte proliferation, increase expression of Ihh/PTHrP, and accelerate terminal differentiation in hypertrophic chondrocytes (solid-line arrow).
BMPs antagonize FGFs at several steps (broken line). Wnt/
-catenin and GH/IGF-1 signaling play vital roles in promoting cell proliferation and inhibiting
chondrogenic differentiation (broken-line arrows).

gether, the effects of IGF-1 on skeletogenesis are not mediated
through osteoprogenitor cells, which is consistent with the hypothe-
sis that IGF-1 exerts its effects on more mature cells of the os-
teoblast lineage.
It has been reported that treatment of cultured chondrocytes
with Dex and IGF-1 repressed IGF-binding protein (IGFBP)-2 ex-
pression without affecting expression of IGFBP-4 and -5. These
chondrocytes proliferated more rapidly than cells treated with IGF-
1 alone. Untreated chondrocytes normally expressed IGFBP-2
through -6, IGF-1 and -2, and their corresponding IGF receptors.
Dex treatment up-regulated expression of IGFBP-5 and IGF-1R and
down-regulated expression of IGFBP-2. However, the inhibitory
effects could be prevented by the addition of the GR antagonist
Org34116 [88]. Therefore, at pharmacological doses, Dex can in-
hibit cell proliferation and alter IGFBP-2, -5 and IGF-1R expres-
sion, suggesting a role for the IGF axis in glucocorticoid-induced
growth retardation.
FUTURE PROSPECTS
In summary, Dex exerts different effects on every stage of em-
bryonic skeletogenesis, the challenge of uncovering the link be-
tween Dex and skeletogenesis is now centered on determining the
mechanisms and molecular signals involved in the activity of Dex
in vitro and in vivo (see Fig. 1). Previously published studies have
provided us with novel insights into the effects of Dex (at pharma-
cological doses) on vertebrate skeletogenesis and have established
the foundation for future functional studies. It is worth noting that
the majority of the studies that have been reported, particularly
those relating to signal transduction and bone growth, have been
conducted in vitro, which should be considered when interpreting
the data. Undoubtedly, elucidation of the molecular pathways in-
volved in this process has been greatly enhanced by the availability
of cell culture models. However, we should not neglect the fact that
Dex exerts a biphasic influence on chondrogenesis and osteoblast
function in vitro and that it can both stimulate and suppress biologi-
cal activity in culture, depending on the Dex concentration used
[53]. There are also other disadvantages of using cell culture sys-
tems. For example, the genetic background of a cell line may have
been transformed such that the results produced may no longer
accurately reflect the in vivo system. Another possibility is that a
cell line may be heterogeneous, among others. Therefore, results
and conclusions drawn from in vitro studies must also be validated
using animal models and/or human bone biopsy studies, particularly
when trying to confirm whether Dex can adversely affect bone
growth. At present, more research will be required to establish a
link between the multiple observed effects of Dex on bone growth.
In addition, it will be useful to discover methods to preserve the
effectiveness of Dex while minimizing its side effects during the
prenatal period.
Dexamethasone Use During Pregnancy
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of
interest.
ACKNOWLEDGEMENTS
This study was supported by “973 Project” (2010CB529703);
NSFC grant (31071054, 30971493) and Guangdong Natural Sci-
ence Foundation (S2011010001593, S2013010013392) to X Yang.
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