Transcriptional Control of Osteogenesis

One of the key issues in studying organogenesis is to understand the mechanisms underlying the differentiation of progenitor cells into more specialized cells of individual tissue. The formation and regeneration of each tissue is associated with a cascade of signals involving a sequential activation of successive genes in response to transcriptional regulators, growth factors, cytokines and hormones. Mediators such as fibroblast growth factor (FGF), transforming growth factor beta (TGF-), insulin-like growth factors (IGF) and many other proteins influence the early stage of cell and tissue formation. The factors are not tissue-specific and control the proliferation and differentiation of most cell types. The proper tissue formation, maturation and function is controlled by factors unique to particular tissue. Transcriptional regulation is the most important step controlling the decision which genes will be expressed at a given time, thus affect the fate of cells. The recent progress in molecular biology, animal models and development of the skeletal phenotype induced by genetic mutations in humans, led to a better understanding of the role of transcriptional factors that govern bone formation and are specifically activated during osteogenesis. The development of the vertebrate skeletal elements may take place through two different mechanisms: osteoblastogenesis and osteochondrogenesis, and relies on the differentiation of the required cell types: osteoblasts and chondrocytes (membranous and endochondral/cartilaginous osteogenesis, respectively), which are derived from common mesenchymal stem cells (Fig. 1.). Osteoblastogenesis (membranous osteogenesis) takes place in the mesenchymal membrane, where osteoblast progenitor cells differentiate directly from embryonic condensed mesenchyme, mature to osteoblasts and begin to secrete type I collagen, proteoglycans and intercellular substance of the organic component of bone – osteoid. These mechanisms are responsible for the development of skull bones, facial bones and parially colarbone. Most of the vertebrate skeleton develops through endochondral ossification, whereby a cartilaginous template is initially formed and subsequently mineralized and replaced bone. Endochondral bone formation (cartilaginous osteogenesis) takes place in long bones starting with the condensation of skeletal precursors, proceeding to the formation of a cartilagenous template. Chondrocytes at the condensation centre stop proliferating and become hypertrophic (enlarged). This process induces differentiation of osteoblast precursors in the perichondrium adjacent to the region of hypertrophic cells. Perichondrial cells adjacent to hypertrophic chondrocytes become osteoblasts, forming bone collar. At the subsequent steps hypertrophic chondrocytes secrete a mineralized matrix and die through apoptosis.


Introduction
One of the key issues in studying organogenesis is to understand the mechanisms underlying the differentiation of progenitor cells into more specialized cells of individual tissue. The formation and regeneration of each tissue is associated with a cascade of signals involving a sequential activation of successive genes in response to transcriptional regulators, growth factors, cytokines and hormones. Mediators such as fibroblast growth factor (FGF), transforming growth factor beta (TGF-), insulin-like growth factors (IGF) and many other proteins influence the early stage of cell and tissue formation. The factors are not tissue-specific and control the proliferation and differentiation of most cell types. The proper tissue formation, maturation and function is controlled by factors unique to particular tissue. Transcriptional regulation is the most important step controlling the decision which genes will be expressed at a given time, thus affect the fate of cells. The recent progress in molecular biology, animal models and development of the skeletal phenotype induced by genetic mutations in humans, led to a better understanding of the role of transcriptional factors that govern bone formation and are specifically activated during osteogenesis. The development of the vertebrate skeletal elements may take place through two different mechanisms: osteoblastogenesis and osteochondrogenesis, and relies on the differentiation of the required cell types: osteoblasts and chondrocytes (membranous and endochondral/cartilaginous osteogenesis, respectively), which are derived from common mesenchymal stem cells (Fig. 1.). Osteoblastogenesis (membranous osteogenesis) takes place in the mesenchymal membrane, where osteoblast progenitor cells differentiate directly from embryonic condensed mesenchyme, mature to osteoblasts and begin to secrete type I collagen, proteoglycans and intercellular substance of the organic component of bone -osteoid. These mechanisms are responsible for the development of skull bones, facial bones and parially colarbone. Most of the vertebrate skeleton develops through endochondral ossification, whereby a cartilaginous template is initially formed and subsequently mineralized and replaced bone. Endochondral bone formation (cartilaginous osteogenesis) takes place in long bones starting with the condensation of skeletal precursors, proceeding to the formation of a cartilagenous template. Chondrocytes at the condensation centre stop proliferating and become hypertrophic (enlarged). This process induces differentiation of osteoblast precursors in the perichondrium adjacent to the region of hypertrophic cells. Perichondrial cells adjacent to hypertrophic chondrocytes become osteoblasts, forming bone collar. At the subsequent steps hypertrophic chondrocytes secrete a mineralized matrix and die through apoptosis. Vascular invasion follows, bringing osteoblast progenitors from the bone collar into the centre of the future bone. Chondrocytes continue to proliferate, lengthening the bone. Osteoblasts of primary spongiosa are precursors of eventual trabecular bone; osteoblasts of bone collar become cortical bone. At the end of the bone, the secondary ossification centre forms through cycles of chondrocyte hypertrophy, vascular invasion and osteoblast activity. A bone marrow expands in marrow space along with stromal cells. Fig. 1. Schematic representation of membranous and endochondral bone formation. Amesenchymal cells condensation and proliferation; B -differentiation; C -production of mature osteoblasts; D -osteoblast differente into osteocytes,blood vessels invade and together with osteoblast start to make bone utilize the mineralized matrix; E -synthesis of extracellular matrix and chondrocytes grow to become hypertrophic; F -apoptosis of hypertrophic chondrocytes and osteoblast invasion; G -vascularization of cartilage and osteoblast maturation, secondary ossification center formation are not shown.
All of phenotypic and physiological changes in mesenchymal cells arise as a result of regulatory events at the genetic level. Mutations or deregulation of these processes lead to skeletal malformation, and/or susceptibility to injury. Over the past years on the basis of in vivo and in vitro studies various transcription factors have been identified that play important roles for skeletal formation being either active in osteoblasts and chondrocytes or in both cell types. The molecular mechanisms controlling the chondrogenic and osteogenic program are becoming elucidated; however, major difficulty in the overall understanding of bone formation is to combine all scattered pieces of information considering the function and importance of individual genes and their products into comprehensive signal network. This chapter presents recent findings on the role of the most important and unique genetic regulators supporting bone formation.

Transcription control of osteoblastogenesis
The specific structure of bone, its function and metabolism are a result of the processes of bone formation, resorption, mineral homeostasis and bone regeneration. Osteoblasts are bone-forming cells that synthesize and mineralize extracellular matrix. In membranous bone formation, they arise from multipotential mesenchymal stem cells under the influence of growth factors, hormones and cytokines. Differentiation of progenitor cells into osteoblast in vitro has been induced by the presence of osteogenic supplements in cultured medium (dexamethasone, ascorbic acid, vitamin D3, β-glycerophosphate). The proper maturation and osteoblast function is directly related to the expression of two key transcription factors of osteoblastogenesis: Runx2 and Osterix.

Runx2 (Runt-related transcription factor 2)
The transcriptional control of the proliferation, growth and differentiation of mesenchymal stem cells into mature bone cells is primarily controlled by Runx2 (also known as CBFA1, AML3 or OSF2). Numerous in vitro studies have shown that Runx2 is a positive regulator of gene expression, whose products are bone extracellular matrix proteins, such as type I collagen, osteopontin, bone sialoprotein and osteocalcin (Komori, 2006). Runx2 is frequently described as the master regulator of osteoblastogenesis. Its deficiency in homozygots leads to different types of bone dysplasia, consisting of genetically determined disorders in the structure of the skeleton (Marie, 2008). Runx2 is also involved in processes related to the maturation of cartilage cells (Makita et al., 2008). However, during dentinogenesis, Runx2 expression is downregulated, and Runx2 inhibits the terminal differentiation of odontoblast. Runx2 expression has also been recently demonstrated in non-skeletal tissues such as breast, brain, sperm and T cells (Leong et al., 2009). Therefore, it may play a role in transmitting epigenetic information encoded in DNA. The mechanism for regulating the expression of genetic information by Runx2 has been intensively studied for many years. Runx2 is a DNAbinding transcriptional factor that interacts with the promoters of specific target genes through the Runt domain. Its potential binding site has been identified at target promoters, for example in the promoter region of sialoproteins between position -84 to -79 and -184 to -179 relative to the transcription start point (Paz et al., 2005). Regions recognized by Runx2 demonstrate a consensus sequence (PuACCPuCa) described as an osteoblast specific element (OSE2). Runx2 belongs to the Runt family. It should be noted that the other two regulators of the Runt family, Runx1 and Runx3, also participate in the induction of osteoblastic genes. Amino acid sequence alignments of all main Runt family proteins are shown in Fig. 2. www.intechopen.com Two distinct mechanisms involving osteoblast-mediated membranous and endochondrial ossification have been proposed: Runx1 participates in membranous bone formation which corresponds to the involvement of this protein in the early stages of osteoblastogenesis, www.intechopen.com while Runx2 plays an exclusive role in both membranous and endochondral bone forming processes that is in the process of osteoblast maturation. Runx3 is engaged in the differentiation of chondrocytes in cartilaginous ossification. The Runt family regulators are proteins that have transcription isoforms. Three isoforms of Runx2 protein are a result of complex process of alternative splicing (Li & Xiao, 2007). However, the specific molecular mechanism involved in the potential isoform functions in bone formation are not well understood. Positive transcription regulators of Runx2 were identified earlier (bone morphogenetic proteins, homeodomain proteins), while the first reports of proteins that inhibit Runx2 gene expression were published in 2009, when the Nieto group showed Snail1 to be a transcriptional repressor at the Runx promoters. Snail1 is a protein involved in the transformation of epithelial cells in the development of embryonic mesenchymal cells (EMT, called the epithelial mesenchymal transition) (De Frutos et al., 2009). Another repressor of Runx2 is Twist -basic helix-loop-helix transcription factor that regulates differentiation of multiple cell types. Twist inhibits osteoblastogenesis by interaction with Runx2-DNA binding domain and in this way prevents its ability to bind DNA (Bialek et al., 2004). Expression of Runx2 is controlled by Foxo1 which can also directly interact with this transcription factors. Teixeira and coworkers suggested that Foxo1 is an early molecular regulator during mesenchymal cell differentiation into osteoblast exerting its effects through regulation and interaction with Runx2 (Teixeira et al., 2010). Several studies have indicated that Runx2 is a context-dependent transcriptional activator and repressor and may interact with other regulatory proteins, suggesting a complex mechanism of osteoblastogenesis control by this factor. So far coactivators of a Runx2dependent transcription include p300 and CBP (CREB-binding protein), which function as transcriptional adapters in interactions with other proteins in multiprotein regulatory complexes. Through direct interaction with Runx2, they up-regulate Runx2-dependent transcription. Runx2 corepressors are components of multiprotein complexes that mediate histone deacetylation and condensation of chromatin, such as the TLE (transducin-like enhancer), mSin3A and HDAC3/4/6. Runx2 interacts with many transcription factors, such as Smad1 and Smad5, Twist, Dlx family, Zfp521. The nature of this cooperation is of great interest to many research teams. The functional activity of Runx2 is particularly sensitive to posttranslational modification of the protein (e.g. phosphorylation, acetylation, methylation). It seems that the preferred interaction of Runx2 with specific cofactors depends on its post-translational modifications (Bae & Lee, 2006). Although Runx2 and its impact on osteoblast differentiation has been widely accepted, our knowledge on the mechanism underlying the process and factors influencing Runx2 expression or activity still require further studies.

Osterix
Osterix (SP7, OSX) is another transcriptional regulator, essential for differentiation of progenitor cells into osteoblasts, and hence for bone formation. This protein belongs to the Sp/XKLF family of transcriptional factors. The common feature of these regulators is the presence of a DNA binding domain, consisting of Cys2His2 zinc fingers. The OSX protein is comprised of 431 amino acids and contains three zinc finger motifs located in the C-terminal part. It is also possible to distinguish a proline-and serine-rich activation domain between 141 and 210 amino acids. Osterix transcription is positively regulated by Runx2, and in Runx2 null mutants, Osterix expression does not occur (Nakashima et al., 2002). It is an osteoblast-specific regulator and its activity has not yet been demonstrated in other cell types. The expression of early markers of osteoblast differentiation (e.g. osteopontin, alkaline phosphatase) in the human osteosarcoma cell line MG63 is not dependent on Osterix (Hatta et al., 2006). In contrast, the activation of late genes, such as osteocalcin, is correlated with the presence of this regulator; thus, it appears that Osterix is a factor required to progress the differentiation of preosteoblasts into mature osteoblasts. Despite its involvement in osteoblast differentiation, Osterix regulatory mechanism is not fully understood yet. Osterix activation region is known to interact with many regulatory proteins, including NFAT (nuclear factor of activated T cell) and primary transcription factor -TF-IIB, or Brg1 -chromatin-remodeling factor. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, novel Osterix-interacting factors have been identified, such as RNA helicase A (RHA) (Amorim et al., 2007). Immunoprecipitation and Western blot analysis has shown Osterix to directly associate with RNA helicase A. Hence, RNA helicase A may act as a component in Osterix regulation of osteoblast differentiation. Osterix activity is regulated by various post-translational modifications including phosphorylation and glycosylation (Chu & Ferro, 2005). It has been shown that calcineurin, a protein phosphatase, affects the function of Osterix through direct interaction and altering its posttranslational phosphorylation form. The application of calcineurin inhibitor resulted in an increasing level of phosphorylated form of Osterix. Nevertheless, it remains unclear how the phosphorylation of Osterix occurs and modulates its function.

Transcriptional control of osteochondrogenesis
In mammals, most skeletal elements are formed through endochondral bone formation, which is characterized by the initial formation of cartilage molds from mesenchymal condensations and their subsequent replacement by bones. Two groups of transcription factors that control the key steps of chondrocyte differentiation have been identified: Sox (sex reversal Y-related high-mobility group box protein) and Runt families. Natural chondrogenesis is initiated by Indian hedgehog signaling and associated with the expression of the major transcription factor Sox9 that controls downstream genes involved in chondrogenesis, and promoting cells to produce cartilage-specific extracellular matrix including collagen type X, collagen type II alpha 1, cartilage oligomeric matrix protein and proteoglycans. In chondrogenesis, Sox9 requires the activity of two other Sox family members: Sox6 and the large isoform of Sox5. Runt family members has also ability to drive chondrocyte differentiation. The stages of chondrocyte differentiation are regulated by a complex series of signaling molecules and transcription factors but also by paracrine factors such as TGF-, BMPs, FGFs, and Wnts.

Sox protein and chondrocyte commitment
Sox9 is considered the master protein that control chondrogenic lineage commitment. Role of Sox9 in chondrogenesis was first demonstrated in a human genetic disease campomelic dysplasia. This disease is caused by heterozygous inactivating mutations in and around the Sox9 gene and is characterized by hypoplasia of all endochondral skeletal elements. Sox9 belongs to a large family of SRY (sex-determination factors) transcriptional regulators which are characterized by a high mobility group DNA-binding domain (HMG-box). This domain preferentially binds CA/TTTGA/TA/T sequence in vitro (Han & Lefebvre, 2008).

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Sox9 binds to DNA within the minor groove of double helix. Chondrocyte-specific genes (i.e.: collagen type II alpha1-Col2a1, collagen type XI alpha2-Col11a2) contain Sox9-binding sites, mutations in this DNA region abolished DNA binding of Sox9 inactivated these gene in vivo and in vitro. Sox9 is required during sequential steps of the chondrocyte differentiation pathway. In skeletal development, Sox9 is expressed during the formation of mature cartilage and mesenchymal condensation in endochondrial ossification except hypertrophic chondrocytes. Abnormal expression of Sox9 leads to severe skeletal disorders like skeletal dysmorphology syndrome or campomelic dysplasia. It was shown in experiments in mouse chimeras using Sox9-double null embryonic stem cells that development of cartilages gene is not carried out without active Sox9. On the other hand, forced expression of Sox9 in non-chondrogenic cells leads to the expression of chondrogenic markers, such as collagen type II alpha1 (Col2a1), aggrecan (Acan) and cartilage oligomeric matrix protein (Comp). Interestingly, overexpre s s i o n o f S o x 9 i n t h e c a r t i l a g e c a u s e s a decrease in chondrocyte proliferation and a delay in bone development. This decrease in proliferation may result from binding of Sox9 to β-catenin, the essential component of the canonical Wnt signaling pathway. Along with the observation, overactivation or deletion of β-catenin in chondrocytes resulted in severe skeletal dysplasias. The expression and function of Sox9 is not restricted to chondrogenesis, suggesting that this regulator may cooperate with other factors in various/different cellular processes. The activity of Sox9 is regulated by a number of intracellular factors. The function of Sox9 may be modulated by phosphorylation cAMP-dependent protein kinase A by parathyroid hormone-related peptide signaling pathway (PTHrP). It is, therefore, possible that Sox9 mediates part of PTHrP action to regulate hypertrophic differentiation (Zhao et al., 2009). Other Sox genes necessary for cartilage formation include Sox5 and Sox6. They are expressed downstream of Sox9 from the prechondrocyte stage and later remain coexpressed with Sox9. Sox5 gene is highly similar to Sox6, and the two proteins share 50% homology with Sox9 in the Sox domain but not in remaining domains. The expression of Sox5 and Sox6 requires Sox9. Roles of Sox5 and Sox6 in chondrogenic differentiation were demonstrated in genetically manipulated mice. Single Sox5 or Sox6 knock-out mice mutants are born with relatively small skeletal defects, whereas double Sox5/Sox6 mutants exhibit a lack of cartilage and endochondral bone formation, becouse Sox5, Sox6, with Sox9 form a trio of transcription factors needed and sufficient for chondrogenesis. Sox5 and Sox6 may form homo-and heterodimers with each other, which bind much more efficiently to pairs of HMG-box binding sites than to single binding sites. Sox5 and Sox6 do not contain any domain that allows for transactivation or -repression of genes transcription and may thus act only to facilitate the organization of transcriptional complexes. Yu Han and Veronique Lefebvre data suggest a new model for the chondrogenic action of the Sox trio: "mesenchymal cells and prechondrocytes express chondrocyte markers at low or undetectable levels because Sox9 has a limited ability to bind to the cartilage enhancers of these genes in the absence of Sox5/Sox6. By inducing Sox5 and Sox6 expression in overtly differentiating chondrocytes, Sox9 gives itself the potential to upregulate its own activity as Sox5/Sox6 now binds to distinct sites on the enhancers and thereby secures Sox9 binding to its recognition site" (Han & Lefebvre, 2008). Only combination of all factors: Sox5, Sox6, and Sox9 successfully induce chondrogenic differentiation in vivo. Chondrogenic system in vitro via the changes in combination of the three Sox protein levels provide a new chondrogenic differentiation model, which may help us to better understand the mechanism of this process, but also adds a powerful tool to cartilage and bone regenerative medicine. www.intechopen.com

Role of Runt family members in osteochondrogenesis
The Runt family of DNA-binding transcription factors regulates cell fate determination in a number of tissues and has been shown to play an essential role in the differentiation of osteoblasts. They play a major role at the late stage of chondrocyte differentiation: in hypertrophic and maturating chondrocytes. In addition to osteoblasts, Runx2 is expressed in the lateral mesoderm, mesenchymal condensations, and chondrocytes. Runx3, which is another Runt family transcription factor, is engaged in the terminal differentiation of chondrocytes (Komori, 2005). Chondrocytes divide and produce a characteristic matrix but then stop dividing, change the matrix they synthesize, and become quite large (hypertrophic). Runx2 and, to a lesser extent, Runx3, are the major transcription factors controlling the crucial steps of osteochondrogenesis. Mice missing Runx2 show a defect in chondrocyte maturation, with lack of hypertrophic chondrocytes in many bones, and mice missing both Runx2 and Runx3 completely lack chondrocytes. The activity of Runx2 and Runx3 is modulated through the interactions with other factors. During chondrogenesis, expression of Runx2, is abolished in cells with heterozygous mutations in and around Sox9 (Akiyama et al., 2005). Dlx5 and Dlx6 proteins cooperate with Runx2 in activation of hypertrophic gene expression as they can physically interact with Runx2 and control chondrocyte hypertrophy in vivo and in vitro (Chin et al., 2007). Karsenty group results suggest that Runx2, in addition to promoting hypertrophy through its expression in chondrocytes, negatively regulates hyperthrophy by acting in the perichondrium thereby providing an additional level of control in order to coordinate chondrocyte maturation and osteoblastogenesis (Hinoi et al., 2006). By using small interfering RNA-mediated knockdown of Bapx1 (also know as Nkx3.2) in cultured chondrocytes an increase in Runx2 expression was shown, what indicate that Bapx1 act as a negative regulator of chondrocyte maturation (Hartman, 2009).

c-Maf protein
Chondrocyte differentiation in the growth plate is an important process for the longitudinal growth of endochondral bones. Sox9 and Runx2 are the most often-studied transcriptional regulators of the chondrocyte differentiation processes, but the importance of additional factors is also becoming apparent. One such factor is c-Maf. It belongs to a subfamily of the basic ZIP (bZIP) transcription factor superfamily, which act as key regulators of tissuespecific gene expression and terminal differentiation in many tissues. This regulator is low expressed in immature proliferating chondrocytes but high expressed in late hypertrophic and terminal chondrocytes, making it a candidate for controlling terminal differentiation. There is increasing evidence that c-Maf and its splicing variant Lc-Maf play a role in chondrocyte differentiation in a temporal-spatial manner. Various types of abnormalities in endochodrnal ossification or chondrocytic differentiation are caused by abnormalities concerning target genes regulated by c-Maf and Lc-Maf as well as lack of c-Maf itself (as phenotype of mice lacking c-Maf show) (Hong et al., 2011). Although the differential expression patterns of c-Maf and Lc-Maf during chondrogenesis were described, the functional differences between them are still unknown. c-Maf can form homodimers as well as heterodimers with other transcription factors. c-Maf binding site was identified: two 13-and 14-base pair palindrome sequences, TGCTGACTCAGCA and TGCTGACGTCAGCA (Kataoka et al., 1994). Studies of the roles of Maf proteins in chondrocyte differentiation and cartilage are under way, as is the identification of genes directly regulated by c-Maf. A causative role for c-Maf will require more hard evidence from direct experiments.

Tissue-nonspecific factors for bone formation
During the development of multicellular organisms, cell fate specification is followed by the sorting of different cell types into distinct domains where the different tissues and organs are formed. It has been shown that the formation and differentiation of tissues and organs during embryogenesis is regulated by the activation of a number of factors, which cannot be considered skeletal specific, although they are thought to play a key role in the differentiation and maturation of the osteoblast phenotype, and were observed in tissues that undergo both membranous and endochondral ossification. Alterations in functions of various other non-bone-specific transcription factors have been also demonstrated to affect osteoblastic differentiation and function. Among the many factors essential for organogenesis, and required for skeletal development are: bone morphogenetic proteins (BMPs), Wingless-type (WNT), homeobox genes HOX/HOM, DLX, MSX, ZPA (regulating the activity of tissue polarity, zone polarizing activity), FGF (fibroblast growth factor), Sonic and Indian Hedgehog (Shh and Ihh, respectively) (Witkowska-Zimny et al., 2010).

Bone morphogenetic proteins
Bone Morphogenetic Proteins (BMPs) belonging to the superfamily of transforming growth factors  (TGF-) are important regulators involved in the differentiation process of forming tissues and organs during embryogenesis, including growth and differentiation of mesenchymal stem cells into osteogenic cells (Phimphilai et al., 2006). BMPs also play a key role in tissue regeneration in the post-embryonic period. Several proteins belonging to the group of BMPs have been described, of which BMP2, BMP4, BMP7 are acknowledged as osteogenic BMPs since they have been demonstrated to induce osteoblast differentiation in a variety of cell types. BMPs, which function by activating intracellular SMAD proteins and kinase signaling cascades (MAP, ERK PI3-K/AKT) are involved in the expression of multiple target genes (Osyczka et al., 2005). BMPs signals directly correspond to the early embryogenesis proteins containing homeodomain (called homeodomain proteins) involved in the development of the skeleton (HoxA10, Dlx3) (Hassan et al., 2006). Furthermore, the transcription factor of early ostoblastogenesis, Runx2, is induced in response to the presence of BMP2, by a SMAD-dependent signal transduction pathway (Phimphilai et al., 2006). Leong and colleagues demonstrated that palmitoylation was involved in the BMP2dependent pathway. The inhibition of palmitoylation reduce osteoblast differentiation and mineralization, but had no effect on cell proliferation (Leong et al., 2009). This study was the first one to show that protein palmitoylation plays an important role in osteoblast differentiation and function. BMPs also play a role in many stages of chondrogenic differentiation, initiating chondroprogenitor cell determination and differentiation of precursors into chondrocytes, and also at the stage of chondrocyte maturation and terminal differentiation (Pizette & Niswander, 2000;Retting et al., 2009). In addition, signalling through the BMP receptors is required for the maintenance of the articular cartilage in postnatal organisms (Rountree et al., 2004). Moreover, BMPs promote cell death and apoptosis of chondrocytes (Zou & Niswander, 1996). Despite numerous studies, the regulatory pathway dependent on BMP is still not fully understood. Although the molecular mechanisms of signal transduction by BMPs are not known, recombinant human BMP2 and BMP7 have been successfully used in clinical applications as a factor assisting the regeneration of bone tissue (Bessa et al., 2008).

WNTs
WNTs are secreted glycoproteins involved in the regulation of embryonic development, as well as in the proliferation and differentiation of many tissues, including bone. WNT signal transmission in the cell occurs via various WNT-dependent pathways, which are always activated by binding WNT proteins to the endothelial Frizzled receptor (Fzd) and its coreceptor (Mbalaviele et al., 2005). Activation of a specific pathway depends on the type of WNT ligand and the current conditions within the cell. Gain-or loss-of-function studies in mice have revealed the function of various component of the pathway. To date 19 WNT ligands and 10 different subtypes of Fzd receptors have been detected. The many players in the WNT cascade hamper the precise elucidation of the mechanism by which WNT signaling specificity is achieved. By far the best characterized cascade is the canonical signaling pathway. It has been reported that binding the WNT to the endothelial Fzd receptor and LRP-5/6 protein (lipoprotein-related protein 5 and 6) on the surface of osteoblast progenitor cells, involves the stabilization of the central player in the canonical WNT pathway --catenin, and regulation of multiple transcription factors. The level of -catenin increases in the cytoplasm, which results in its transport to the nucleus and activation ofosteoblast differentiation genes expression. This process is mediated by transcriptional factors, including Runx2 and Osterix. An appropriate level of the canonical Wnt signalling is crucial for chondrogenesis, demonstrated by the abnormal growth plate phenotype in mice harbouring inactivated β-catenin in chondrocytes (Ryu et al., 2002). -catenin is highly expressed in mesenchymal cells committed to the chondrocytic lineage but down-regulated at the stage of early chondrogenic differentiation, upon up-regulation of Sox9 (Akiyama et al., 2004). Sox9 interacts with -catenin and enhances its phosphorylation and subsequent degradation. Wnt signalling is again up-regulated during hypertrophy and promotes chondrocyte hypertrophy and endochondral ossification (Hill et al., 2005). WNT/-catenin signaling is also important for mechanotransduction and fracture healing (Westendorf et al., 2004;Chen et al., 2007).

HOX -homeobox proteins
HOX protein family, encoded by a subclass of homebox genes, belongs to the regulators controlling the process of embryogenesis in vertebrates. These homeobox transcriptional factors are capable of binding to specific nucleotide sequences on DNA where they either activate or repress genes. The expression of the HOX genes in the developing area is temporally and spatially dynamic. They are critical for proper formation of skeletal tissue. HoxA and HoxD serve in a dose-dependent manner to regulate the size of specific cartilage elements. A surprising finding was that loss of these genes does not interfere the chondrocyte proliferation and differentation, but the growth of the individual elements is not established properly. Therefore, in controlling osteochondrogenesis they act later to regulate longitudinal growth of skeletal elements (Boulet & Capecchi, 2004). HoxA10 and other homeobox genes responsible inter alia for osteoblastogenesis also participate in the regulation of cell proliferation, differentiation and maturation of osteoblasts in the process of modeling and regeneration of bone tissue in adult organism (Zakany et al., 2007). Research conducted in the last two years has shown the dependence of Runx2 gene expression and Runx2-dependent genes (encoding osteocalcin, alkaline phosphatase, bone sialoprotein) on HoxA10. It also showed that HoxA10, both directly and independently of Runx2, regulates the transcription of certain genes during www.intechopen.com osteoblastogenesis. Two mechanisms of HoxA10 action have been proposed: as a component of the BMP2 signaling cascade, prior to Runx2 involvement in the induction of genes as a factor osteoblastogenesis, and as a chromatin modifier in the promoter regions of genes specific to bone tissue. Combinatory mechanisms are operative for a regulated transcription of osteoblast genes through the diversification of sequence-specific activators and repressors that contribute to patterns of gene expression and the multistep process of programming involved in bone formation.

DLX -Distal-less homeodomain proteins
DLX is a family of transcription regulators containing the homeobox domain, which are activated by a BMP2 signal. Dlx3 expression is synchronized with the stages of osteoblast growth and induced by BMP2 (Hassan et al., 2006). An overexpression of Dlx3 in osteoblast progenitor cells changes the expression of the differentiation markers: type I collagen, osteocalcin and alkaline phosphatase. It has been demonstrated that two members of this family, Dlx3 and Dlx5, up-regulate the endogenous expression of Runx2. Like HoxA10, Dlx3 and Dlx5 may participate in ostoblastogenesis through the activation of Runx gene expression, but also directly through other genes, independently of Runx2. It has been demonstrated that Dlx3 and Dlx5 regulate the synthesis of Runx2, but at different stages of the osteoblast differentiation process: Dlx3 in the early stages of osteoblastogenesis, while Dlx5 in mature osteoblasts (Hassan et al., 2009). DLX proteins may bind to Runx2 promoter region, but only after the removal of another homeobox protein, MSX (mesh-less homeodomain), which acts as a repressor. Dlx3 and Dlx5 binding sites next to Runx2 binding site have been identified in the promoter region of alkaline phosphatase and osteocalcin genes. However, it has been shown that the process of bone tissue differentiation occurs in mutants without the Dlx5 gene. This suggests that the Dlx5 protein acts as a regulator of expression in the multiprotein activation complex and not as the main transcription activator of genes involved in the differentiation of the osteogenic lineage (Samee et al., 2008). The specific regulation mechanism of Runt gene expression, alongside with other Runx2-dependent genes with the participation of several classes of homeotic genes, has been suggested in a few works by Jane B. Lian team (Hassan et al., 2006(Hassan et al., , 2009.

MSX proteins
Vertebrate Msx are homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene. The mammalian Msx gene family consists of three members, named Msx1, Msx2, and Msx3. Msx3 is expressed only in the dorsal neural tube, whereas Msx1 and Msx2 are widely expressed in many organs during embryonic development. Msx proteins interact with other homeodomain proteins to regulate transcription. Heterodimers formed between Msx and other homeodomain proteins such as Dlx2, Dlx5, Lhx2 and Pax3 result in mutual functional antagonism in vitro. Msx1 and Msx2 are among the critical factors involved in osteoblastogenesis. Null mutation of Msx2 leads to a number of defects in the construction of the skeleton especially in craniofacial region. Loss-of-function and gain-of-function studies show that in mice as in humans Msx1 and Msx2 are required for normal craniofacial morphogenesis (Alappat et al., 2003). They play a role in crucial processes during limb morphogenesis. Mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. A number of data have shown that Msx genes are downstream targets of BMP signaling in the limb. At early stages, Msx1 and Msx2 are expressed in nearly identical patterns that overlap significantly with BMP2, BMP4 and BMP7. An antagonistic role of Msx2 has been demonstrated in relation to Dlx5 during osteoblast proliferation and differentiation. Dlx5 is activated in the later stages of osteoblastogenesis, which correlate with increasing levels of proteins characteristic of terminally differentiated osteoblasts, such as osteocalcin, while Msx2 adversely affect these processes. On the basis of these studies, it has been suggested that Msx2 stimulates the process of cell proliferation and inhibits cell differentiation. Several models for the Msx2 and Dlx5 relationship have been proposed. In the first model, Runx2-Msx2 forms a complex that deactivates expression of Runx2 and Runx2-dependent genes. With the increasing levels of Dlx5, a Dlx5-Msx2 complex is formed and free Runx2 protein can in consequence activate specific genes. In the second model for Dlx5 and Msx2 interaction, proteins compete for binding to common binding sites in the promoter region of specific genes and they also regulate each other at the transcriptional level. In both cases a balance between the levels of Msx2 and Dlx5 may be critical for osteoprogenitor cell proliferation and differentiation (Lallemand et al., 2005). It is certain that, Msx with other morphogenes and transcription factors build a complex cellular network that control the development and behavior of cells. It would be of great interest to identify the direct target genes of Msx proteins in vivo and their associated cellular processes including proliferation, apoptosis, cell adhesion and migration during organogenesis.

Hedgehog proteins
Hedgehog (Hh) is evolutionarily conserved family in vertebrates, which include Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehogs that control numerous aspects of development: cell growth, survival, and differentiation, and pattern almost every aspect of the vertebrate body plan. Dhh expression is largely restricted to gonads. Ihh is specifically expressed in a limited number of tissues, including primitive endoderm, gut and prehypertrophic chondrocytes in the growth plates of bones. Shh is the most broadly expressed mammalian Hh signaling molecule. During early vertebrate embryogenesis, Shh expressed in midline tissues affects skeletal development and most epithelial tissues. The use of a single morphogen for such a wide variety of functions is possible because cellular responses to Hh depend on the type of responding cell, the amount of Hh received, and the time cells are exposed to it (Varjosalo & Taipale, 2008). During skeletogenesis, Shh and Ihh provide positional information and initiate or maintain cellular differentiation programs regulating the formation of cartilage and bone. Malfunction of the Hh signaling network can cause severe skeletal disorders.

Sonic hedgehog
Shh signaling acts to initiate an osteogenic program of mesenchymal cells. The human Shh has three exons that encodes a 462 amino acid polypeptide. The protein is synthesized as a precursor molecule that undergoes cleavage of a signal peptide and then autoproteolytic cleavage. This reaction mediated by cholesterol leads to a 19 kDa N-terminal product (Shh-N) with the signalling domain and a C-terminal product of 25 kDa (Shh-C) possessing the cleavage domain closely associated with cholesterol transferase activity. The Shh is highly conserved among vertebrates. For example, there is 92.4% identity between human and mouse Shh proteins. Increased Hh signaling promotes osteogenesis in various bone-forming www.intechopen.com cells in vitro. The stimulatory action of Shh on osteogenic differentiation was already reported in few studies, suggesting a close interaction between Shh and BMP-2 or parathyroid hormone-related peptide (Yuasa et al., 2002.) Shh has been shown to play a key role in patterning of the limb, i.e. misregulation of Shh results in severe limb abnormalities. Hence, Shh is required for proper ZPA (regulating the activity of tissue polarity, zone polarizing activity) signaling and anterior/posterior limb formation (Hill, 2007;Towers et al., 2008, James et al., 2010.

Indian hedgehog
Growth and differentiation of the endochondral skeleton relies on a complex interplay among different signaling factors to regulate the orderly morphogenesis of the skeleton. Among these, Ihh appears to play a central role in coordinating chondrocyte proliferation, chondrocyte differentiation and osteoblast differentiation. During endochondral bone development, Ihh is synthesized by chondrocytes leaving the proliferative pool (prehypertrophic chondrocytes) and by early hypertrophic chondrocytes. Ihh is a master regulator of both chondrocyte and osteoblast differentiation during endochondral bone formation. Ihh mutants show: (i) reduced chondrocyte proliferation; (ii) initially delayed, then abnormal chondrocyte maturation, and (iii) absence of mature osteoblasts. St-Jacques and coworkers suggest a model in which Ihh coordinates diverse aspects of skeletal morphogenesis through parathyroid hormone-related peptide dependent and independent processes (St-Jacques et al., 1999). Mouse Ihh cDNA encodes a 411 amino acids polypeptide with a predicted 27 amino acids signal peptide. After the post-translational modification arises a 19 kDa lipid-modified protein. At the cell surface, Ihh activity is mediated by binding to the transmembrane receptor, and signaling through the transmembrane Gprotein coupled receptor. Hedgehogs, including Ihh are important signaling molecules during embryonic development and are highly conserved within and across species. Mouse and human Ihh share 100% amino acid identity of the signaling domain.

Zinc finger protein: PLZF and Zfp521
Zinc finger proteins are believed to be one of the most common classes of proteins in humans (approx. 3-4% of human genes encode proteins containing zinc finger domains). One of previously described osteoblastogenesis factor -Osterix also belong to this protein type. Promyelocytic leukaemia zinc finger protein (Zinc finger protein 145, PLZF) belongs to the family of Krüppel-like zinc finger proteins. It is a transcriptional repressor involved in cell cycle control and has been implicated in limb development, differentiation of myeloid cells, and spermatogenesis. So far little is known about the regulation of PLZF expression. PLZF is one of the highly expressed genes during in vitro osteoblastic differentiation in many human cell types. Small interfering RNA-mediated gene silencing of PLZF results in a reduction of osteoblast-specific genes expression such as alkaline phosphatase, collagen type 1, osteocalcin and even Runx2 genes. These findings indicate that PLZF plays important roles in early osteoblastic differentiation as an upstream regulator of Runx2. Because the expression of PLZF was unaffected by the addition of bone morphogenetic protein 2 in vitro, it may indicate that PLZF acts independently of the BMP signaling pathway (Ikeda et al., 2005). The molecular pathways by which PLZF exerts its function in bone formation are still under investigation.
Another regulator from Kruppel-like zinc finger protein family associated with osteogenesis is Zfp521. Zfp521 is expressed in osteoblast precursors, osteoblasts and osteocytes, as well as chondrocytes. Forced expression of Zfp521 in osteoblasts in vivo increases bone formation and bone mass. In contrast, overexpression of Zfp521 in vitro antagonizes, while knockdown favors, osteoblast differentiation and nodule formation. Zfp521 binds to Runx2, repressing its transcriptional activity (Wu et al., 2009). The balance between Zfp521 and Runx2 may therefore contribute to the regulation of osteoblast differentiation and bone formation.

MicroRNAs in skeletal development
One of the first steps in understanding of cell determination requires ascertaining which particular genes are activated in a particular cell, either temporarily or continuously. Microarrays technology are used as a tool for quantitative expression analysis of many gene transcripts in parallel as well as study expression of small non-coding microRNAs that repress mRNA translation and thereby regulate differentiation and development. Posttranscriptional regulation by non-coding RNA molecules has been discovered to be an important mechanism to control cellular differentiation, also during bone formation. An increasing number of miRNAs have been identified to regulate osteoblast differentiation. They promote bone formation by targeting negative regulators of osteogenesis or negatively regulate osteoblastogenesis by targeting important osteogenic factors. Among many miRNA negatively regulating osteogenesis are Runx2-targeting miRNAs: miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, andmiR-338 (Zhang et al., 2011a, 2011b). They significantly impede osteoblast differentiation, and their effects can be reversed by the corresponding anti-miRNAs. Only a few miRNAs have been identified to specifically regulate chondrogenesis and cartilage homeostasis: miR-140 and miR-199 as negative regulators and miR-675 as a positive regulator (Miyaki et al., 2009;). miR-675, whose expression is upregulated by Sox9, positively regulates chondrocyte specific gene i.e. Col2a1, and in this way promotes chondrogenesis (Dudek et al., 2010;Lin et al., 2009).

Conclusion
Bone is a highly dynamic tissue, which is regulated by tissue-specific transcription factors, as well as by the number of homeotic genes, active both during the organization of tissue and organs in the embryonic period as well as in mature bone. The most important factors for osteogenesis are compiled and briefly overview in the Table 1. Transcriptional regulators control the expression of target genes by the interaction with cofactors, coactivators, chromatin remodelling complexes and finally with the general transcriptional machinery. Their participation in the regulation of bone formation process is complex and require further experimental work to provide understanding of their role and elucidate interactions with other factors of signal cascades. Cellular balance between various regulatory proteins is extremely important. Many studies have been conducted using murine or human cell lines, which are often tumor cell lines e.g. MG63 -human osteosarcoma line. The regulatory pathways and routes of signal transduction in these experimental systems may not correspond to those occurring in healthy human bone cells. Therefore, it is important to enhance our knowledge about proliferation, differentiation and regeneration of bone based on in vitro and in vivo studies of normal human cells in the www.intechopen.com context of natural tissue. From a biomedical point of view, identifying an important regulators of bone formation and regeneration in humans has raised the possibility that manipulating its expression, function, or signalling pathway could have a major therapeutic impact in identifying new targets and opening new avenues for the treatment of bone diseases, such as osteoporosis.