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. Author manuscript; available in PMC: 2013 Jul 29.

Genetic and molecular control of Osterix in skeletal formation

Krishna M Sinha1,*,Xin Zhou1,1
1Department of Endocrine Neoplasia and Hormonal Disorders, UT MD Anderson Cancer, Center Houston, TX, U.S.A. 77030
*

Correspondence:ksinha@mdanderson.org, Ph: +1 713-563-9117

1

Department of Genetics, UT MD Anderson Cancer Center

PMCID: PMC3725781  NIHMSID: NIHMS490700  PMID:23225263
The publisher's version of this article is available atJ Cell Biochem

Abstract

Osteoblast differentiation is a multi-step process where mesenchymal cells differentiate into osteoblast lineage cells including osteocytes. Osterix (Osx) is an osteoblast-specific transcription factor which activates a repertoire of genes during differentiation of preosteoblasts into mature osteoblasts and osteocytes. The essential role of Osx in the genetic program of bone formation and in bone homeostasis is well established.Osx mutant embryos do not form bone and fail to express osteoblast-specific marker genes. Inactivation ofOsx in mice after birth causes multiple skeletal phenotypes including lack of new bone formation, absence of resorption of mineralized cartilage, and defects in osteocyte maturation and function. Since Osx is a major effector in skeletal formation, studies on Osx gained momentum over the last five-seven years and implicated its important function in tooth formation as well as in healing of bone fractures. This review outlines mouse genetic studies that establish the essential role of Osx in bone and tooth formation as well as in healing of bone fractures. We also discuss the recent advances in regulation ofOsx expression which is under control of a transcriptional network, signaling pathways, and epigenetic regulation. Finally we summarize important findings on the positive and negative regulation of Osx’s transcriptional activity through protein-protein interactions in expression of its target genes during osteoblast differentiation. In particular, the identification of the histone demethylase NO66 as an Osx-interacting protein which negatively regulates Osx activity opens further avenues in studying epigenetic control of Osx target genes during differentiation and maturation of osteoblasts.

Keywords: Osterix, Osx, NO66, gene expression, osteoblasts, osteocytes

Introduction

Bone formation occurs through two developmental processes – intramembranous and endochondral ossification. Intramembraneous ossification which mainly forms craniofacial bone is mediated by the differentiation of the condensed mesenchyme into osteoblasts. In endochondral ossification, mesenchymal cells in the condensation differentiate into chondrocytes, forming a cartilage template that is later replaced by bone [Fang and Hall, 1997]. In skeletogenesis, Sox9, Runx2 and Osterix are three essential transcription factors that have important roles in the cell-fate decision process by which mesenchymal cells become chondrocytes and osteoblasts, through activation of cell type-specific genes. Sox9 is the earliest expressed transcription factor in prechondrogenic mesenchymes, which differentiate into chondrocytes. Runt-domain-containing Runx2, another early differentiation factor, is expressed in the condensed mesenchymes.Runx2-expressing cells segregate from osteochondroprogenitors to form precursor osteoblasts. Expression ofOsx inRunx2-expressing precursors induces these cells to differentiate into mature and functional osteoblasts, and finally into osteocytes during bone formation [Karsenty, 2001;Nakashima and de Crombrugghe, 2003;Nakashima et al., 2002;Nishio et al., 2006;Yang and Karsenty, 2002;Zhou et al., 2010;Zou et al., 2006]. Several other transcription regulators including Wnt/β-catenin, homeodomain protein Dlx, Msx2, ATF4, and bone morphogenetic proteins have been known to play important roles in controlling gene expression during differentiation and maturation of osteoblasts [Hassan et al., 2009;Karsenty, 2008;Rodda and McMahon, 2006;Yang et al., 2004]. In this review we will primarily focus on the role of Osx in control of genetic and molecular programs of osteoblast differentiation

1. Control of Osx in the genetic program of bone formation

Over a decade ago, the lab of deCrombrugghe identified Osterix (Osx/sp7) cDNA using a differential hybridization approach from BMP2-treated and untreated C2C12 cells [Nakashima et al., 2002]. BMP2 treatment induces differentiation of C2C12 cells into osteoblasts by stimulating expression of osteoblast genes. Molecular characterization of this cDNA led to the identification of Osx, which contains a proline-rich activation domain at the N-terminus spanning aa 1–289 and three C2H2-type zinc finger DNA-binding domains at the C-terminus. In DNA transfection studies, Osx containing aa 1–172 is sufficient for transcriptional activation. Osterix is sometimes referred to as Sp7 because of the striking homology with zinc finger DNA-binding domains of Sp1, Sp3 and Sp4 transcription factors. During mouse embryonic development,Osx expression begins around E13.5 in calvaria and perichondrium of long bones and is robustly expressed at E15.5 in all osseus elements.Osx is also expressed at a lower level in pre- and hypertrophic chondrocytes. It is clear that Runx2 is required to prime the prechondrogenic mesenchyme segregate into the precursor osteoblast lineage, whereas Osx is subsequently required to accomplish the osteoblast differentiation pathway (seeFigure 1).

Figure 1. Osteoblast differentiation is a multi-step process.

Figure 1

Multipotent mesenchymal precursors in condensed mesenchymes differentiate intoSox9-expressing osteochondroprogenitors, which segregate into chondrocytes that express high levels of Sox9, and preosteoblasts that express high levels ofRunx2 and low level ofCol1a1 andAkp. Osx expression is needed to accomplish the differentiation of preosteoblasts to mature and functional osteoblasts that express high levels of osteoblast markers includingCol1a1, Akp, Bsp, andOc. Osx controls the genetic program of osteoblast differentiation (shown in light blue area). Functional osteoblasts produce extracellular matrix molecules which form a calcified bone matrix.Osx-null cells are arrested in their differentiation and acquire chondrocytes phenotype by expressing chrondrocyte markers. The molecular control of Osx is depicted in light yellow area. Expression ofOsx mRNA and protein is positively regulated by signaling pathways (BMP2, MAPK, Igf1), Runx2, Dlx5, ER-stress pathway (IRE1a/XBP1), and negatively regulated by DNA methylation and microRNAs, p53, and TNF. The transcriptional activity of Osx is documented by NFATc as positive regulator and by NO66 histone demethylase as negative regulator.

Osx-null cells are arrested in their differentiation

Osx-null embryos either do not express osteoblast markers or express them at very low levels, despite the normal expression ofRunx2. Expression ofCol1a1, which is expressed at higher levels in mesenchyme of wild-type mice, is significantly reduced inOsx mutants. Other osteoblast markers includingOsteonectin, Osteopontin andBonesialo protein are undetectable in endochondral and membranous skeletal elements inOsx mutants.Osteocalcin, the very late and specific osteoblast marker normally expressed at E18.5, is also absent from prefigured skeletal zones inOsx-null embryos. Expression ofRunx2 is also normal in the condensed mesenchyme of membranous and endochondral skeletal zones inOsx-null embryos and long bones, butOsx is evidently not expressed in skeletal parts ofRunx2-null embryos, indicating that Runx2 is an upstream regulator ofOsx. These studies strongly indicate that Osx triggersRunx2-precursors to develop into osteoblasts by activating expression of osteoblasts genes [Nakashima et al., 2002].

Is Osx a negative regulator of chondrocytes?

Osx-null embryos, unlikeOsx-expressing embryos, do not form bone but apparently chondrocyte differentiation and cartilage formation are not affected in null embryos.Runx2-positiveOsx-null cells express chondrocyte-specific genes such asSox9 andCol2a1 and acquire a chondrocyte-type cell fate [Nakashima et al., 2002]. These observations lead to speculation that Osx inhibits chrondrocyte differentiation inRunx2-expressing precursor osteoblasts.Runx2 is expressed early on in prechondrogenic mesenchyme cells, which express high levels ofSox9. Later on,Osx expression is stimulated,Runx2-positive cells exit as precursor osteoblasts andSox9 expression is down-regulated, and thereby suggesting that expression ofSox9 andOsx is mutually exclusive. ThoseRunx2/Osx-expressing cells strictly differentiate into mature and functional osteoblasts in whichSox9 is not expressed. TheRunx2-expressing precursors, prior toOsx expression, remain in the chondrogenic lineage and do not progress into the preosteoblast stage. The spatial and temporal expression ofOsx, studied byKaback et al (2008), also indicates that the increase inOsx expression specifically coincides with the onset and progression of osteoblast differentiation, but occurs after the maturation of chondrocytes [Kaback et al., 2008]. In addition, the levels ofOsx mRNA remain high after cartilage is formed and remodeled into bone. These studies suggest that Osx plays dual roles in inhibition of chondrocyte formation and in promoting osteoblast differentiation (Table 1).

Table 1.

Following publications are suggested for further information on Osterix studies

Description of studiesExperimental modelsAuthors
Discovery & function of Osx in osteoblast differentiation and bone formation during embryonic developmentmouse KO studiesNakashima et al (2002)
Role of Osx in bone homeostasis in adult and in formation of osteocytesConditional mouse KOZhou et al (2010)
Frameshift mutation inOSX geneOsteogenesis imperfectaLapunzina P et al (2010)
Function of Osx in cementogenesis (tooth formation)Conditional mouse KOCao et al (2012)
Expression patterns of Osx during tooth formationMouse studiesHirata et al (2009)
Osx-mediated inhibition in differentiation and maturation of chondrocytesMouse and cell cultureKaback et al (2008)
Runx2 independent activation of Osx through Dlx5Cell culture studiesLee et al (2003)
Runx2-mediated activation of OsxCell culture studiesNishio et al (2006)
Requirement of precursor osteoblast (Osx+) in primary ossification as well as in bone healing processMouse studies using lineage tracingMaes et al (2010)
ER-stress response in activation of Osx during osteoblast differentiationCell culture studiesTohmonda et al (2011)
Function of Osx in bone-related injuriesMouse studiesTu et al (2006,2007)
Inhibition of bone formation by p53 through negative regulation of OsxMouse KO studiesWang et al (2006)
Inhibition of osteoblast differentiation by NO66 histone demethylase through interactions with OsxCell culture studiesSinha et al (2010)
p38-mediated Osx phosphorylationCell culture studiesOrtuno et al (2010)
BMP-2 dependent and independent activation of Osx through MAPKCell culture studiesCelil et al (2005)Celil & Cambell (2005)
Akt and Erk1/2 dependent Osx activityCell culture studiesChoi et al (2011)
mir-93 controls Osx proteinCell culture studiesYang et al (2012)

Essential role of Osterix in bone homeostasis in adults

Osx also plays an essential role in formation of adult bones and in expression of osteoblast genes in the adult. Zhou et al. demonstrated that inactivation ofOsx in adult mice, using LoxP/inducible Cre activity, causes multiple skeletal phenotypes including lack of new bone formation, abnormal cartilage accumulation beneath the growth plate, and defects in osteocyte maturation and function. In addition to down-regulation of osteoblast genes inOsx-ablated adult mice, expression of osteocyte-specific marker genes includingSost, Dkk1, Dmp1, Phe is decreased. These studies reveal a new role of Osx in resorption of cartilage matrix eventually replaced by bone matrix. Note that this strategy was used to deleteOsx in all cell types usingpCMV Cre-ERT2 [Zhou et al., 2010]. Role of Osx in degradation of cartilage matrix was further implicated by another study indicating that Osx plays important role in calcification of cartilaginous matrix by directly targeting activation MMP-13 in the hypertrophic chrondrocytes of the growth plate during endochrondral bone formation. Conditional and global ablation of Osx caused the loss of MMP-13 expression and arrest of endochondral ossification at hypertrophy stage [Nishimura et al., 2012]. WhenOsx is inactivated specifically in osteoblasts using 2.3kbCol1a1-Cre during embryonic development, no apparent skeletal defects were observed in new-born mice. However, in growing adult stage mice 8 weeks old or older,Osx mutant mice displayed reduced bone volume in trabecular and lumbar vertebra and decreases in thickness and number of trabecular bone, thus exhibiting human skeletal defects similar to osteopenia [Baek et al., 2009]. Further, inactivation ofOsx in osteoblasts of adult mice with inducible Cre recombinase driven byCol1a1-Cre ERT2 also causes such skeletal phenotypes as reduced expression of osteoblast genes, reduced osteoblast differentiation and function, and mild reduction in bone loss [Baek et al., 2010]. Taken together, these studies depending upon time ofOsx inactivation clearly indicate that Osx continues to play a role as an anabolic regulator that supports the homeostasis of bone in adults.

Although ablation ofOsx in mice causes inhibition of bone formation, transgenic mice that over-expressOsx by2.3kb Col1a1 promoter show osteopenia, characterized by woven bone structure in the cortical bone, as well as decreased expression of osteoblast genes, especiallyOc. InOsx-expressing transgenic mice, osteoblast differentiation is inhibited and immature osteoblasts are accumulated. These results indicate that proper dosages of Osx are required to promote osteoblast differentiation and bone formation in adults [Yoshida et al., 2012].

2. Mutations inOsx are associated with human skeletal diseases

Genetic analyses using GWAS, SNP, and linkage mapping are used to identify genes that cause human diseases. Skeletal disorders such as osteoporosis and osteogenesis imperfecta (OI) are associated with mutations in causative genes, including type I collagen gene, which causes low bone mass, and bone mineral density and makes the animal susceptible to multiple bone fractures. Lapunzina et al identified a homozygous single nucleotide deletion (1052delA) mutation inOSX gene of an Egyptian child who had been clinically diagnosed with OI. This patient had repeated fractures, mild bone deformities, delayed tooth eruption etc.OSX mutation occurred at the third zinc finger of the DNA-binding domain, causing a frameshift mutation that produced a truncated OSX protein. The reduced activity of OSX may have altered expression of the OSX target bone-specific genes includingtype1 collagen in this patient [Lapunzina et al., 2010]. Genome-wide association studies further revealed a number of genetic variants associated with bone mineral density (BMD) and osteoporosis. Among several GWAS-derived loci,OSX is the only locus containing genetic variants over-represented in female childhood obesity for increased BMD. Further SNPs in theOSX region indicate a strong correlation between genetic variants at theOSX locus with adult lumbar spine and BMD [Timpson et al., 2009;Zhao et al., 2011]. These genetic, clinical and epidemiological studies indicate the significance of Osx for overall bone health in adults. Further studies are required to identify mutations in theOSX gene from patients with bone disorders to solidify the correlation between Osx mutations and bone diseases.

3. Osx is needed for odontoblast differentiation and tooth development

The process of tooth formation is quite similar to that of bone formation. Dental mesenchyme cells differentiate into odontoblasts, which produce and secrete collagenous and non-collagenous matrix protein during dentin formation. Dentin sialo phospho protein is a major component of non-collagenous dentin matrix protein and essential for dentin formation. Mouse genetic and biochemical studies demonstrated that both Runx2 and Osx have important roles in tooth formation and function independently in activation of specific genes required for odontoblast differentiation [Chen et al., 2009;Hirata et al., 2009;Zhao et al., 2007]. Chen et al showed that expression ofDsp is positively correlated with that ofOsx, but not withRunx2. Runx2 andOsx expression overlap in alveolar bone osteoblasts, odontoblast, amyloblasts, and dental pulp cells in E16 mouse embryos, whenDsp is weakly expressed in preodontoblast. By the later stage of differentiating and mature odontoblasts at E18, expression ofRunx2 dramatically decreases, whereasOsx expression remains intense in odontoblast and dental pulp cells in whichDsp expression is strongly expressed. It appears that Osx is needed for differentiation and maturation of preodontoblast to odontoblast through activation of specific late marker genes. This situation of Osx function is reminiscent of osteoblast differentiation where Osx is needed for differentiation of preosteoblast to osteoblasts. Overlapping expression ofOsx andDsp is also observed at the postnatal stage of tooth development. Forced expression ofOsx in mouse odontoblast-like cells stimulates expression ofDsp mRNA, indicating that theDsp gene could be a direct target of Osx activation [Chen et al., 2009]. Future studies such as RNASeq in combination with ChIP sequencing are required to validate indications that Osx directly activates expression of odontoblast genes during tooth formation.

In the last five years Osx has generated much attention in bone as well as dental research. Recently, Cao et al used mouse models to elucidate for the first time Osx’s role in the formation of cellular cementum – a mineralized tissue that supports tooth development. Transgenic mice that overexpress Osx driven by the 3.6kbCol1a1 display an increase in cementum formation, whereas tissue and stage-specific deletion ofOsx in mice2.3 Col1a1-Cre in embryos or withCMV-Cre ERT2 in adults showed sharp declines in cementum formation and expression ofDmp1. These data further support the important function of Osx in tooth formation [Cao et al., 2012].

4. Potential use of Osterix in bone fracture and repair

Given that Osterix is a classic osteoblast marker and essential for bone formation, exploitation of Osx as an anabolic molecule is an appealing avenue in the repair of bone fractures and injuries. Bone repair is a complex process that includes spatial and temporal expression of multiple molecules and interactions of these molecules in formation of new bone during fracture healing. A review on gene therapy approaches in bone regeneration with recent works on molecules that are required for bone healing is summarized [Franceschi, 2005]. Studies involving controlled bone injuries followed by healing in animals have demonstrated thatOsx is expressed intensely during regeneration of bone cells at fracture sites. Implanting ofOsx-expressing BMSc (bone marrow stroma cells) at the surgically incisor of rat calvaria accelerates bone healing at the injured calvaria much more than implanted control BMS cells by forming new bone at the injured calvaria as well as by inducing expression of bone-specific genes [Tu et al., 2007]. Further,Osx-expressing BMSc cells implanted into nude mice also induced bone formation through acceleration in osseo integration of implants [Tu et al., 2006;Xu et al., 2009]. Kaback et al showed that in mice with controlled bone fracture, the fracture callus and cartilage began to form at day 7 after fracture and continued at day 10 with an increase inSox9 mRNA.Osx was expressed in the osteoblast of the woven bone of the fracture callus at day 10, but was excluded from cortical bone, hypertrophic chondrocytes and blood vessels. At day 14 post-fracture,Osx was mostly expressed in osteoblasts surrounding the healed sites whereas expression ofSox9 decreased and cartilage of the callus remodeled into bone [Kaback et al., 2008]. In the same study, treatment of PTHrP or BMP2 to osteochondro progenitors which were derived from mouse limb buds that expressed high levels ofSox9 induced expression ofOsx but PTHrP or BMP2 decreased that of the chondrocyte markersSox 9 andCol2a1. Over-expression ofOsx in these progenitors decreasedSox9 level, suggesting that Osx might inhibit chondrocyte differentiation while promoting osteoblast differentiation [Kaback et al., 2008].

The mechanism of migration of precursor osteoblasts at the primary ossification site during normal bone formation or fracture healing was further demonstrated by lineage tracing approach using two mouse strains, harboring Osx-CreERt and Col1a1-ERt transgenes, and crossing these mice with Rosa26R-LacZ reporter [Clarkin and Olsen, 2010;Maes et al., 2010]. Injection of 4-OH tamoxifen to transgenic mice at specific time during development activates expression of Cre recombinase which subsequently inducesLacZ expression [Maes et al., 2010]. In this study, authors reported that two populations of osteoblast-lineage cells exist at the periochondrium; the precursor osteoblasts which areOsx-positive, and the differentiated osteoblasts which areCol1a1-positive. These experiments showed that Osx/LacZ expressing precursor osteoblasts were sparsely present in the cortex but were abundantly present inside the trabecular bone whereas Col1a1/LacZ cells were enriched at the cortex and engaged into cortical bone formation, and were hardly present in the trabecular bone. This study proposes a mechanism by which precursor osteoblasts (Osx-/LacZ positive cells) enter at the site of primary ossification center along with blood vessels becauseOsx-expressing precursors are intimately associated with endothelial cells of blood vessels. Additionally, bone fracture studies performed in Osx-Cre:GFP mice that allowed tracing of GFP positive cells further revealed that before fracture these Osx-GFP positive cells were present in the thin periosteal layer of the cortical bone, but after fracture these cells along with blood vessels were massively localized to the woven bone callus at the healing regions. These studies indicate the potential application of Osx as bone regenerative medicine in healing of bone fractures.

5. Osx expression is dependent on or independent of Runx2 activity

InOsx-null embryosRunx2 is expressed whereas inRunx2-null embryos,Osx is not expressed, and therefore, Osx can be placed downstream of Runx2, where it activatesOsx expression (seeFigure). Sequence analysis of theOsx promoter reveals that several putative Sox9, VDRE, Runx2, Dlx5 and Sp1 response elements are present within upstream regulatory sequence of theOsx gene [Nishio et al., 2006]. In a reporter assay, Runx2 was able to activate theOsx promoter through direct interactions with Runx2-binding element present within 1 kb of theOsx promoter. Other studies also indicated thatOsx expression is not exclusively dependent on Runx2 activity. InRunx2-null calvarial cells in whichOsx is normally not expressed, BMP2 treatment inducedOsx expression through upregulation of Dlx5, which interacted withOsx promoter containing Dlx5 binding elements [Lee et al., 2003]. BMP signaling did not directly induce expression ofOsx but through phosphorylation of Dlx5 enabled the interactions of Dlx5 with theOsx promoter. These studies indicate thatOsx expression is mediated through alternate pathways independently of Runx2. It would be reasonable to conclude that, although Runx2 alone may not be sufficient forOsx expression, it functions in tandem with other signaling pathways to mediate Runx2-independent Osx expression during osteoblast formation. Interestingly, Osx regulates its own expression through a feedback mechanism by interacting with its own promoter [Yoshida et al., 2012]

Osx expression under control of signaling pathways

Several studies elucidated the role of bone morphogenetic protein 2 (BMP2) and insulin-like growth factor (Igf1) signaling pathways in activation of osteoblast genes during osteoblast differentiation. These growth factor-ligand complexes elicit a cascade of signal transduction pathways followed by phosphorylation of downstream substrates to control gene transcription [Huang et al., 2007;Jadlowiec et al., 2004]. BMP2 is known to induceOsx expression in a Runx2-dependent manner. BMP2/Smad pathway targets activation ofRunx2, which in turn activatesOsx expression. Igf1 also up-regulatesOsx expression without alteringRunx2 expression. Although Igf1 response inOsx expression is not as strong as BMP2, a combination of BMP2/Igf1 synergistically increasesOsx expression in human mesenchymal stem cells (hMSC) compared to Igf1 alone. This suggests thatOsx expression requires other signaling pathways beyond BMP2/Smad/Runx2. The synergistic activation ofOsx expression is most likely due to common convergent mitogen-activated protein kinase (MAPK) and protein kinase D (PKD) pathways, activated by the BMP2/Igf1 signaling [Celil and Campbell, 2005;Celil et al., 2005]. MAPK has been reported to participate in induction of Alp activity and Ca++ deposition during bone formation. There are four members of the MAPK super family--extracellular-signal-regulated kinase 1/2 (Erk1/2), c-Jun amino-terminal kinase (JNK), p38, and Erk5. BMP2 treatment of hMSC activates MAPK components p38 and Erk1/2 through phosphorylation of p38 and Erk1/2, which mediate BMP2-inducedOsx expression. Inhibition of p38 and Erk1/2 signaling using specific inhibitors down-regulates BMP2-mediatedOsx expression. Further over-expression of dominant-negative form of p38 or Erk in hMSC completely abolishedOsx expression under osteogenic media, thereby strongly indicating thatOsx expression involves the MAPK pathway rather than BMP2/Smad/Runx2 signaling, because MAP/Erk does not inhibitRunx2 expression. However, activated MAP/Erk does phosphorylate Runx2 to increase the stability, and the transcriptional activity of Runx2 in expression ofOsx. A recent report further suggests a novel mechanism ofOsx expression during osteoblast differentiation under osteogenic media by ascorbate-dependent prolyl hydroxylase domain protein (PHD) and proteasome pathway, which targets degradation of yet-to-be-defined transcriptional repressors of theOsx gene [Xing et al., 2011]. Ascorbate effects inOsx expression are independent of BMP2, Igf1, ECM-mediated integrin signaling, and MAPK pathways, because blockade of either pathway does not affectOsx expression. However, inhibition of PHD by the specific inhibitors dimethyloxallyl glycine and ethyl 3, 4-dihydroxybenzoate strongly affects ascorbate-inducedOsx expression and OB differentiation.

Function of tumor-suppressor p53 has also been documented in osteoblast differentiation and bone formation by regulatingRunx2/Osx, supported by both gain of function and loss of functions in mouse models [Liu and Li, 2010]. Wang et al showed that tumor-suppressor p53 is able to inhibitOsx expression in DNA transfection assays as well as in vivo using mouse genetic approaches [Wang et al., 2006]. Inhibition by p53 ofOsx expression does not occur through direct binding on theOsx promoter, because there is no p53 binding sequence present in theOsx gene. It was suggested that like other target genes, p53 repressesOsx expression by preventing recruitment of p300 and other activators to the target chromatins. Inp53-null mice, expression ofOsx, but notRunx2, is up-regulated and so are the other Osx target genes. As a result, the loss of p53 induces accelerated osteoblast differentiation and high bone mass phenotype inp53 mutant mice[Wang et al., 2006].

ER-stress responds toOsx induction

Mature osteoblasts produce high levels of extracellular matrix (ECM) proteins during skeletal development, often resulting in accumulation of misfolded proteins that cause endoplasmic reticulum stress (ER stress) [Boot-Handford and Briggs, 2010;Murakami et al., 2009;Park et al., 2012]. To circumvent the ER stress so that ECM proteins may be secreted by osteoblasts, the unfolded protein response pathway is activated by ER stress transducers including IRE1α (inositol-requiring endonuclease 1α), which is involved in processing of transcription factorXBP1 mRNA [Tohmonda et al., 2011]. XBP1 then binds to UPR elements and activates many genes to alleviate ER stress. TheOsx promoter also contains the recognition sequence for XBP1 and UPR. Tohmonda et al showed that upon treatment of thapsigargin, an ER stress inducer, theOsx gene is activated by XBP1 through interaction withOsx promoter during BMP-2-induced osteoblast differentiation of mouse embryonic fibroblasts [Tohmonda et al., 2011]. Overexpression ofXBP1 stimulatesOsx expression but not Runx2, indicating a functional role of the IRE1a-XBP1 pathway inOsx expression during differentiation of preosteoblasts to mature osteoblasts.

Negative regulation ofOsx expression

Epidermal-growth factor receptor (EGFR) and tumor-necrosis factor (TNF) oppose osteoblast differentiation by negatively regulating expression of osteoblast genes. EGFR inhibitsRunx2 expression and consequentlyOsx expression through stimulation of HDAC levels in cells [Zhu et al., 2011]. Several HDACs including HDAC 3–7 are known to inhibit osteoblast differentiation by inhibiting Runx2 activity [Bradley et al.;Bradley et al., 2011;Jensen et al., 2007;Jensen et al., 2008;Kang et al., 2005]. TNFα promotes bone loss and delayed osteoblast differentiation through upregulation of Prx1 (paired related homeodomain). Prx1 interacts with theOsx promoter and repressesOsx expression [Lu et al., 2011]. Characterization ofOsx gene revealed that two transcription start sites, each driven by independent promoters, give rise to two isoforms ofOsx; Osx1 being the more abundantly expressed form compared to alternatively splicedOsx2. TNF-response elements are present in both promoters, and the binding of TNF at theOsx promoter inhibitsOsx expression in preosteoblasts MC3T3 cells.

Epigenetic control ofOsx expression by DNA methylation and miRNAs

DNA methylation is a repressive epigenetic mark that is associated with silencing of the key genes required for cellular differentiation [Minard et al., 2009;Ong and Corces, 2012]. Demethylation of those key genes is essential for activation of gene expression that allows cell-type differentiation during development. A study using cell line based experiments reported that the promoters of the osteoblast genes includingDlx5,Osx,Oc andAlp are hypomethylated in osteoblast cells that express these genes and are hypermethylated in non-osteogenic cells, which do not express these genes [OLee et al., 2006]. Treatment with azacytidine (5Aza), an inhibitor of DNA methylation, activates expression of silenced osteoblast genes includingOsx andDlx5 in C2C12 mesenchymal cells. This suggests that DNA methylation restricts expression of those genes to prevent the differentiation of multipotent precursors into specific cell lineages [Hupkes et al., 2011]. Hupkes et al further reported that the majority of 5Aza-treated C2C12 cells differentiate into myoblasts and to a lesser extent into osteoblasts because 5Aza treatment stimulated expression of the key transcription factorsMyoD andOsx through DNA demethylation [Hupkes et al., 2011]. It is likely that hypermethylation of theOsx promoter plays a crucial role in imposing a barrier in expression of this gene in pluripotent mesenchymes. Hence, demethylation of theOsx promoter might begin to occur in precursor osteoblasts, which results in restriction of its expression to osteoblasts. Along this line, a recent report indicates that an osteocyte-specific markerSost gene is hypermethylated in osteoblasts that do not expressSost gene. In osteocytes, theSost promoter is hypomethylated and is expressed [Delgado-Calle et al., 2012]. Note that Osx is needed for the maturation and function of osteocytes by activating osteocyte-specificSost, Dmp1, andPhex genes [Zhou et al., 2010]. We should speculate that DNA methylation ofOsx and its target genes is lost at specific developmental stages to ensure transcription of these genes in osteoblasts and osteocytes during bone formation.

MicroRNAs are small noncoding RNAs that negatively regulate gene expression at post-transcriptional and post-translational level by directly targeting mRNA of the key genes, and then subsequently control cellular activities including proliferation and differentiation. A comprehensive review on the role of several miRNAs in skeletal development recently appeared in literature [Lian et al., 2012]. Among those miRNAs, miR-93,125, 135, 138, 637 have been reported to function in regulation of Osx and to inhibit osteoblast formation [Eskildsen et al., 2011;Goettsch et al., 2011;Schaap-Oziemlak et al., 2010;Yang et al., 2012]. Yang et al showed that lentivrus-mediated overexpression of miR-93 suppressed osteoblast differentiation and mineral deposition through inhibition of Osx protein, but not Osx mRNA, by directly binding to coding region ofOsx gene. Interestingly, this study also revealed that expression of miR-93 gene is under the control of Osx activity because Osx interacted with the promoter of miR-93 gene and overexpression of Osx suppressed miR-93 expression. These data indicate that that miR-93 regulates differentiation program of osteoblast through Osx/Sp7 feedback mechanism [Yang et al., 2012]. Another miR-637 is overexpressed in adipocytes and suppressed Osx expression in these cells by directly targeting the 3UTR of theOsx gene. Thus, miR-637 promotes transdifferentiation of mesenchymes cells into adipocytes by inhibiting Osx expression and then subsequent osteoblast differentiation [Zhang et al., 2011]. Others miRNA-125b, 135b and 138 have also been reported to negatively regulate Osx expression directly or indirectly in non-osteoblasts such as vascular smooth muscle cells, unrestricted somatic cells or human stromal mesenchymal cells, all of which have potential to differentiate into osteoblasts in which these miRNAs are down-regulated.

6. Control of Osx transcriptional activity

Given that Osx was discovered over a decade ago and Osx is a recognized key regulator of bone formation, we are dismayed that we have such limited knowledge of the molecular mechanisms of Osx activity in activation of osteoblast genes. In this section, we will update our knowledge from recent studies of the regulation of Osx activity.

NFTAc, TFII, p300 and Brg1 promote Osx activity

Nuclear factor of activated T cells (NFATc), an essential transcription factor for osteoclast differentiation, also plays important role in osteoblast differentiation through positive regulation of Osx activity. NFATc induces Osx-dependent activity of its target promoter 2.3kbCol1a1 in reporter assays. NFATc interacts with the activation domain of Osx and this interaction is needed for recruitment of NFATC at the Osx-targetCol1a1 promoter. In Osx knocked-down cells (by siRNA), occupancy of NFATc at theCol1a1 promoter is sharply decreased. Further, there is no activation of the 2.3kbCol1a1-reporter inNFATc−/− primary osteoblasts, suggesting that NFATc acts positively in osteoblast differentiation by stimulating Osx activity [Koga et al., 2005]. Other study shows that the Osx fragment with aa 141–210 is sufficient for activation and interacts with TFII and that the C-terminus of Osx (280–428aa) interacts with Brg1, a catalytic component of SWI/SNF complexes of ATP-dependent chromatin remodeling activity [Hatta et al., 2006].

NO66 histone demethylase inhibits Osx activity

NO66 is a jumonji C containing protein identified as Osx-interacting polypeptides using a proteomic approach and mass spectrometry. NO66 exhibits demethylase activityin vitro with dual specificity for lysine 4 and 36 of histone H3 [Sinha et al., 2010]. NO66 is a part of the pre-ribosome complex, localized mainly in nucleoli but also in nucleoplasm, ubiquitously expressed in all tissues, and evolutionarily conserved among vertebrates [Eilbracht et al., 2004]. Histone demethylases control the levels of histone methylation in chromatin and regulate the transcriptional state of the gene in various cellular activities including proliferation, differentiation, senescence etc [Benevolenskaya, 2007;Bernstein et al., 2006;Cloos et al., 2008;Klose et al., 2006;Martin and Zhang, 2005;Tsukada et al., 2006]. Along these lines, the interactions of NO66 demethylase with Osx should be considered physiologically significant in regulating osteoblast differentiation through modulation of Osx activity [Sinha et al., 2010].

Interactions between Osx and NO66 occur through the activation domain of Osx and JmjC domain, including the C-terminus of NO66, in vitro and in vivo. In DNA transfection assays, NO66 inhibits the Osx-dependent activation of the Osx-targetBsp andOc promoters. Knock-down of NO66 by shRNA in preosteoblast MC3T3 cells accelerates osteoblast differentiation in these cells by stimulating bothAlp activity and extracellular matrix deposition. Expression ofCol1a1,Bsp andOc genes is significantly up-regulated in NO66-depleted MC3T3 cells. NO66 inhibits expression of Osx target osteoblast genes likely through interaction with Osx and through regulating histone methylation levels in the chromatin of these genes. Chromatin immunoprecipitation (ChIP) assays indicate that there is an inverse relationship between occupancy of NO66 histone demethylase, and that of Osx as well as H3K4me3 or H3K36me3 [Sinha et al., 2010]. This supports the hypothesis that interactions of NO66 with the chromatin cause demethylation of these histone lysines for the maintenance of gene repression in the preosteoblast state. These studies open new insights into the regulation of osteoblast genes through epigenetic mechanisms involving histone methylation of the osteoblast-specific chromatin and provide clear evidence for a physiological role of NO66 in osteoblasts.

7. Phosphorylation of Osx regulates its transcriptional activity

Post-translational modification by phosphorylation elicited by signaling pathways is a key cellular event in controlling the activities of transcription factors during gene activation. Numerous studies have implicated for phosphorylation of Osx triggered by signaling pathways such as BM2 and MAPK in the regulation of Osx activity will be discussed here. Osx is phosphorylated by p38 MAPK at Ser-73 and Ser-77 to increase protein stability and to interact with transcriptional activators including p300 at the targetBsp andFmod genes during osteoblast differentiation. BMP-2-induced Osx or ectopically expressed Osx exists as doublet polypeptide bands due to phosphorylation. Inhibition of p38 MAPK inhibits Osx expression at both mRNA and protein level [Ortuno et al., 2010]. Wang et al showed that Spata4 (spermatogenesis associated 4 gene) interacts with Erk1/2 in osteoblasts and enhances phosphorylation of Erk1/2, which then phosphorylates Osx to increase protein stability and activity of Osx during activation of Osx target genes [Wang et al., 2011].

Based on converging evidence, phosphorylation of Osx is indispensible for Osx activity for expression of osteoblast genes. Akt is a serine/threonine-specific protein kinase that phosphorylates Osx at threonine residues present within Akt recognition element RXXS/T near the C-terminus of Osx. BMP-2 treatment of C2C12 cells induces Osx protein in an Akt-dependent manner. Further, Akt stimulates Osx-dependent activation of theOc,Alp andBsp promoters in transfection assays, indicating that phophorylation by Akt increases protein stability and Osx activity during osteoblast differentiation [Choi et al., 2011]. Control of Osx expression and the transcriptional activity of Osx are outlined in the figure.

8. Concluding remarks

In this review, we have compiled many of the important research findings in genetic and molecular control of Osx in bone and tooth formation. Osx is a major effector and essential for activation of bone-specific genes that support bone formation in embryos and adults. Runx2 is a critical in formation of precursor osteoblasts from mesenchymes and Osx is a critical for differentiation ofRunx2-expressing precursors into mature and functional osteoblasts. Since Osx is a classic osteoblast marker, the question remains to be addressed whether Osx once expressed takes control of transcriptional program of osteoblast target genes independently of Runx2 function. Animal models and cell culture studies indicate the potential for therapeutic use of Osx in bone injury repair. The published literature we have reviewed gives a brief assessment of Osx’s role in control of bone formation, but unlike Runx2 which has been extensively characterized till date, we do not have explicit knowledge of the mechanisms underlying Osx expression or the regulation of Osx activity, which is essential for osteoblast formation. A list of suggested articles on detailed study of Osx is included in this review for the reader. Osx and its interactions with other proteins and post-translational modifications by phosphorylation and methylation, signaling pathways, miRNAs, and DNA methylation, any and all of which may regulate Osx expression and activity, should be considered for future work to better understand how Osx controls chromatin structure of its target genes during osteoblast differentiation. Finally we leave a big question to our readers and for future studies – is Osx a slave of Runx2 or the master of skeletalogenesis?

Acknowledgments

We extremely thank Dr. Benoit deCrombrugghe, Dept. of Genetics, M.D. Anderson Cancer Center, Houston, TX, for his unstinted support to us in conducting studies on Osterix, and his valuable suggestions during preparation of this review. Author’s work was supported by grant AR049072 (B. deCrombrugghe) and AR061590 (KS).

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