Dexamethasone Use During Pregnancy: Potential ...

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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 skeletogenesis. 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 periods. 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 numerous 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 signaling 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 osteogenic cell differentiation and mesenchymal cell lineage commitment. 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 longterm 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 depending on how frequently (e.g., single or repeated dose) glucocorticoids are administered [2-7]. On the other hand, a fetus is normally 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 treatment 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 effects 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: [email protected] Tel: +86-20-85220254, E-mail: [email protected]

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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 hyperplasia, premature delivery caused by pregnancy-induced hypertension (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 longterm 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 premature infants to prevent prematurity-related complications and to treat neonatal respiratory distress syndrome. Despite this, Dex remains 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 adolescents 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 exposure 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 ossification – 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 osteoblasts. However, the vertebrate skeleton primarily develops via © 2014 Bentham Science Publishers

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endochondral ossification, which is a well-orchestrated and intricately 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, osteogenesis occurs not only in growing bones but also throughout adult life, and it is involved in the formation of primary and secondary 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 during 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 preventing prematurity-related complications. It has been reported that exposure to Dex during early gestational stages can cause minor cranial-skeletal abnormalities, including encephalocele and meningocele, 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. Neural crest cells also contribute to the formation of the palate, and animal models have been developed that use hydrocortisone, prednisolone and Dex on pregnant to produce embryos with cleft palates. 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 animal 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 inhibited 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 expression is lost sometime between the middle and late neurula

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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 number of somite pairs produced when embryos were treated with Dex. As somites differentiate to form the sclerotome, which will eventually form the vertebrae, it was therefore not surprising that Dex treatment led to malformations of the axial skeleton. The crownrump 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 proteins (BMPs) expression observed following GR-morpholino treatment. Further molecular analysis identified multiple putative glucocorticoid 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 conditions 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 decreases 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 peripherally by soft connective tissue. Limb myogenic cells, which originate 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

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particular, the signaling genes involved in this process are Shh, Hox, Fgf10, Fgf8, Tbx4, Tbx5 and the BMPs, which must be expressed 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 development. THE REGULATORY EFFECTS OF DEXAMETHASONE ON DIFFERENT STAGES OF BONE DEVELOPMENT Endochondral ossification is composed of two crucial morphogenetic 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 microvasculature, 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 chondrogenesis 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 longitudinal bone growth. More specifically, Dex exposure significantly reduces cell number, cell proliferation, and proteoglycan synthesis and increases alkaline phosphatase (ALP) activity during the chondrogenesis in the ATDC5 cell line, which mimics the in vivo process 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 microenvironment 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 extracellular matrix (ECM)-related, metabolic, cytokine and growthfactor 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 proliferation decreases; the chondrocytes become hypertrophic; the ECM surrounding the hypertrophic chondrocytes becomes progressively mineralized; and finally, the mineralized matrix serves as a template 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 exposure. A comparative microarray analysis was conducted on mouse embryonic chondrocytes treated with a pharmacological dose (10-7

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M) of Dex. It appears that Dex acts on cartilage by regulating multiple 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 developmental 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 determining 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 regulation 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 developmental biologists are how undifferentiated precursor cells acquire spatial patterning information, and in turn, how these cells differentiate and respond to the information provided. The developing vertebrate limb has been extensively studied as a model for addressing these questions, and as a consequence, the body of knowledge generated by such studies has provided novel insights into the mechanisms 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 remains 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 importance 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 malformations. These findings are summarized here, and the important features 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 multifunctional roles in specifying the dorsal-ventral axis embryos and in determining cell fate at the neural plate and during limb bud development [49]. BMPs induce a variety of biological activities in an assortment of cell types at various stages of differentiation, although 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 progenitor 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 controlled to yield a balance between proliferation and terminal differentiation during endochondral ossification. BMPs exert profound effects on chondrocyte maturation, and BMP-2 and BMP-6 are


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 explant 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 different drug concentrations used or the specific culturing systems and manipulation protocols. INDIAN HEDGEHOG AND PARATHYROID HORMONERELATED PROTEINS During endochondral ossification, the Indian hedgehog (Ihh) and parathyroid hormone-related (PTHrP) proteins have been identified as components of the negative feedback loop that regulates chondrocyte hypertrophic differentiation. Ihh is a dominant regulator of bone development and is involved in coordinating chondrocyte proliferation and differentiation, as well as osteoblast differentiation. This protein directly stimulates chondrocyte proliferation and determines the length of the proliferating chondrocyte columns through the stimulation of PTHrP synthesis. Furthermore, Ihh expression also defines the sites where hypertrophic differentiation begins. In the cartilage growth plate, the biological activities of mature chondrocytes and early hypertrophic chondrocytes are precisely 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 development, 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 Gli1independent 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 differentiation. In vitro analysis revealed that glucocorticoids repressed 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 Dexinduced development. Studies involving transgenic mice that specifically overexpressed 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 reversed 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 onset of hypertrophic differentiation [58].

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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 undoubtedly 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 chondrocytes and in the perichondrium. FGFR2 is expressed in the chondrogenic condensation, perichondrium, periosteum and primary spongiosa. FGFR3 is expressed in proliferating chondrocytes. Finally, FGF-7, -8 -17 and -18 are expressed in the perichondrium [60]. Each of these growth factors appears to play distinct and important 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 differentiation. Dex is routinely added to MSC cultures to promote cell proliferation and cell pellet expansion, and these effects can be enhanced when combined with a low concentration of FGF-2 (1 ng/mL) [6567]. Dex enhances chondrogenesis and osteogenesis in pellet cultures by increasing nodule formation, glycosaminoglycan deposition, collagen type II synthesis and SOX9 expression. It also positively promotes cell maturation by increasing expression of the developmental markers STRO-1 and ALP [67, 68]. MSCs expanded with Dex/FGF-2 display lower ALP levels than Dexexpanded cells but significantly higher ALP levels than FGF-2expanded 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 prolonged endochondral bone growth. This phenotype is accompanied by the expansion of proliferating chondrocyte columns and hypertrophic 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 ossification, 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 associated 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 chondrocyte 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 independently of Ihh and PTHrP. BMPs can oppose the effects of FGF signaling by increasing chondrocyte proliferation and inducing Ihh expression. BMPs and FGFs have opposing effects on terminal


hypertrophic chondrocyte differentiation, and these pathways can be considered as antagonistic at several levels [74]. In this context, chondrocyte proliferation, osteoblast differentiation and hypertrophic differentiation are the result of antagonistic interactions between 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 expressed 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/-Catenindependent and -Catenin-independent pathways. It has been reported that Wnt/-Catenin signaling was down-regulated in a Dexinduced cleft palate mouse embryo model and that the downstream molecules of Wnt/-catenin signaling, including -catenin, Lef-1, cJun and phospho-c-Jun, were completely inhibited by Dex treatment. Indeed, altered signaling in the affected cells directly impaired their ability to form a normal palate [28]. In the limb bud, Wnt ligands secreted by the ectoderm promote the underlying mesenchymal 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 skeletogenesis and glucocorticoids [76-78]. Micro-CT scans of Col2.311HSD2 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) accumulation within osteoblasts, chondrocytes and mesenchymal progenitors [78]. These observations suggest that intramembranous ossification 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 mesenchymal cell fate from osteoblasts to chondroblasts resulted in suture abnormalities, suggesting that ectopic endochondral ossification 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 tendency of these cells to form osteoblasts. This effect could be blocked by the introduction sFRP1, a Wnt inhibitor, to the co-

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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 progenitor 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 influencing 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 osteoblastogenetic potential. The up-regulation of ALP expression induced by Wnt-3a could be effectively suppressed through the combined activity of osteogenic supplements, Dex and 1,25dihydroxyvitamin 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 upregulated 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 hormone 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 interactions between glucocorticoids, GH and parathyroid hormone (PTH) have recently been investigated in detail. In growing chondrocytes 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 primary 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 glucocorticoids 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 significantly increased the length of hypertrophic zone, whereas Dex alone had no significant effect [86]. Treatment of osteogenic precursors 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-


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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 chondrocytesand the process of bone ossification , although the effects are variable and may be dependent 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 hypothesis that IGF-1 exerts its effects on more mature cells of the osteoblast lineage. It has been reported that treatment of cultured chondrocytes with Dex and IGF-1 repressed IGF-binding protein (IGFBP)-2 expression without affecting expression of IGFBP-4 and -5. These chondrocytes proliferated more rapidly than cells treated with IGF1 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 inhibit cell proliferation and alter IGFBP-2, -5 and IGF-1R expression, suggesting a role for the IGF axis in glucocorticoid-induced growth retardation. FUTURE PROSPECTS In summary, Dex exerts different effects on every stage of embryonic skeletogenesis, the challenge of uncovering the link between 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 involved 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 biological activity in culture, depending on the Dex concentration used [53]. There are also other disadvantages of using cell culture systems. 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.


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 Science Foundation (S2011010001593, S2013010013392) to X Yang. REFERENCES [1] [2] [3]

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Received: December 11, 2013

Accepted: February 4, 2014

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