Minireview: Transcriptional Regulation in Development of Bone
http://www.100md.com
内分泌学杂志 2005年第3期
Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Henry Kronenberg, Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114.
Abstract
Regulation of gene expression by transcription factors is one of the major mechanisms for controlling cellular functions. Recent advances in genetic manipulation of model animals has allowed the study of the roles of various genes and their products in physiological settings and has demonstrated the importance of specific transcription factors in bone development. Three lineages of bone cells, chondrocytes, osteoblasts, and osteoclasts, develop and differentiate according to their distinct developmental programs. These cells go through multiple differentiation stages, which are often regulated by specific transcription factors. In this minireview, we will discuss selected transcription factors that have been demonstrated to critically affect bone cell development. Further study of these molecules will lead to deeper understanding in mechanisms that govern development of bone.
BONE DEVELOPMENT IS achieved through the use of two distinct pathways, intramembranous and endochondral bone formation. In intramembranous bone formation, mesenchymal cells condense and directly differentiate into bone-forming osteoblasts. In contrast, in endochondral bone formation, mesenchymal cells condense and then become chondrocytes. This cartilage mold then directs the formation of osteoblasts, which form mature bone. This bone is continually remodeled by cycles of bone formation and resorption. Osteoclasts, of hematopoietic origin, are responsible for bone resorption. Thus, three distinct cell types (chondrocytes, osteoblasts, and osteoclasts) from two cell lineages direct the formation and remodeling of bone (1).
Endochondral Bone Formation and Chondrocyte Differentiation
Formation of the cartilage anlage starts with mesenchymal condensation (2). The molecular mechanism that regulates this process at the transcriptional level is not fully understood. However, because bone morphogenetic protein (BMP) signaling appears crucial for this process (3), Smads [a name derived from Sma and MADD (Mother against decapentaplegic)], transcription factors downstream of BMP signaling (4), likely play an important role. After the formation of mesenchymal condensations, cells start proliferating again and differentiate into chondrocytes (Fig. 1).
FIG. 1. Differentiation of bone cells of three lineages and its regulation by transcription factors. Transcription factors important for bone cell differentiation are indicated by bold black letters with arrows at major differentiation steps. Signaling molecules are indicated by blue letters. Osteoblasts and chondrocytes are derived from common mesenchymal precursors. During endochondral bone formation, mesenchymal cells condense and differentiate into chondrocytes. BMP signaling is likely required for these processes. Sox9 and its related molecules L-Sox5 and Sox6 play a pivotal role in commitment and maintenance of chondrocyte phenotypes. In the growth plate, chondrocytes proliferate and further differentiate into column-forming cells, then into postmitotic hypertrophic chondrocytes. Runx2 expression stimulates terminal differentiation. Runx2 and Osx are both required for osteoblast differentiation. Runx2 expression starts before that of Osx. Its activity is regulated by interaction with other transcription factors such as members of the Twist family. Osteoclasts differentiate from hematopoietic lineage cells through multiple steps. M-CSF and RANLK are essential external stimuli for osteoclastogenesis. PU.1, MITFs, Nf-B, Fos/Fra1, and NFATc1 are all required for differentiation of mature osteoclasts.
Sox9 (sex reversal Y-related high-mobility group box protein) and chondrocyte commitment
Analysis of genetically manipulated mice demonstrated that Sox transcription factors, members of the high mobility group superfamily, are necessary for conversion of condensed mesenchymal cells to chondrocytes. Studies of chimeric mice (5) and conditional knockouts (6) have shown that Sox9 is required for formation of normal mesenchymal condensations, for conversion of mesenchymal cells to chondrocytes, for proliferation of chondrocytes, and for suppression of premature conversion of these chondrocytes to hypertrophic chondrocytes. Sox9 is also required for the production of L-Sox5 and Sox6, two related Sox family members required for normal chondrocyte function (7). Sox9 is not only required for development of chondrocytes but also directly regulates expression of genes important for chondrocyte function, such as Col2a1 (8), Col11a2 (9), Agc1 (aggrecan) (10), and Mia (Cdrap) (11). The function of Sox9 may be modulated by phosphorylation by the PTH-related peptide signaling pathway (12). It is, therefore, possible that Sox9 mediates part of PTH-related peptide action to regulate hypertrophic differentiation.
Expression of Sox9 begins in the mesenchymal condensation. BMPs can induce SOX9 expression, and noggin, a BMP antagonist, blocks expression of SOX9 in mesenchymal condensations (13). Cytokines such as IL-1 and TNF-, and nuclear factor B (NF-B), the transcription factor that mediates many actions of these cytokines, suppress Sox9 expression, partly through induction of posttranscriptional mRNA degradation (14, 15); this suppression of SOX9 probably contributes to chondrocyte loss in inflammatory arthritis. How specific transcription factors regulate Sox9 gene transcription is a central question, although it appears complicated; genetic analysis of human and mouse Sox9 mutations demonstrated that cis elements quite distant from the transcription start site of the Sox9 gene are critical for Sox9 transcription in cartilage (16).
Interestingly, overexpression of Sox9 in the cartilage causes a decrease in chondrocyte proliferation and a delay in bone development. This decrease in proliferation may result from binding of Sox9 to ?-catenin, the essential component of the canonical Wnt (a name that combines wingless and int) signaling pathway (17). Some Wnt members are expressed in cartilage, and effects of Wnt signaling in cartilage have been demonstrated (18, 19, 20, 21). Along with the observation that overactivation or deletion of ?-catenin in chondrocytes resulted in severe skeletal dysplasias, these findings suggest that the transcription complex, lymphoid enhancer-binding factor (LEF)/T-cell factor (TCF)/?-catenin, regulates some aspects of cartilage development (17).
Runx2 and chondrocyte hypertrophy
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 because chondrocytes stop dividing and become hypertrophic. Runx transcription factors are members of the runt family, named for the DNA binding domain, conserved across species from Drosophila to humans (22). Mice missing Runx2 show a defect in chondrocyte maturation, with lack of hypertrophic chondrocytes in many bones (23), and mice missing both Runx2 and Runx3 completely lack chondrocytes (24). Furthermore, overexpression of Runx2 hastens hypertrophy and even converts chondrocytes in the trachea, which normally never hypertrophy, into hypertrophic chondrocytes and then bone (25, 26).
The stages of chondrocyte differentiation are regulated by a complex series of signaling molecules and transcription factors in addition to Sox9 and Runx2/3. Differentiation of early periarticular chondrocytes into flat, columnar proliferating chondrocytes appears to be regulated by multiple signaling molecules such as Indian hedgehog (Ihh) and Wnts (19, 27). It is, therefore, likely that some of the downstream transcription factors triggered by Ihh and wnts, Gli family members, and the ?-catenin/TCF/LEF complex, play a role in this step. Chondrocyte differentiation is accompanied by changes in proliferation. Alterations in fibroblast growth factor (FGF) signaling (28, 29), BMP signaling (30), and Ihh signaling (31, 32, 33) participate in regulating chondrocyte proliferation. The suppressive effect of fibroblast growth factor signaling on proliferation is regulated by signal transducer and activator of transcription-1 at the transcriptional level (34). Overexpression of Ihh is associated with an increase in cyclin D1 expression (33), which ultimately stimulates cell cycle progression by activating E2F transcription factors (35). BMP signaling, therefore, involving Smad 1, 5, and 8 transcription factors, generally stimulates hypertrophic differentiation (30, 36, 37, 38, 39). Misexpression of a homeobox-containing transcription factor, Dlx5, which is normally expressed in the prehypertrophic region of the growth plate, stimulates hypertrophic differentiation in developing chicken limbs (40). Considering that Dlx5 is downstream of BMP signaling in several other cell types, Dlx5 and its related molecule, Dlx6 (41), may mediate BMP action regulating hypertrophic differentiation. These animal models with Dlx modulation, however, need cautious interpretation because neither misexpression nor deletion of the genes is chondrocyte specific.
Osteoblast Differentiation
Two transcription factors, Runx2 containing a runt domain (42, 43) and Osterix (Osx; SP7) with a zinc-finger motif (44) are absolutely required for osteoblast differentiation during both intramembranous and endochondral bone formation. Analysis of Osx null mice shows that Osx is genetically downstream of Runx2. Runx2 is expressed in the lateral mesoderm, mesenchymal condensations, and chondrocytes in addition to osteoblasts. Both overexpression of Runx2 and expression of a dominant-negative form of Runx2 in osteoblasts impairs bone formation, suggesting that regulation of different stages of osteoblast differentiation by Runx2 is complex (45, 46). Runx2 is regulated by phosphorylation and also interacts with other transcription factors, such as Smads 1 and 5 (47, 48), Smad 3 (49), signal transducer and activator of transcription-1 (50), Menin (51), Hey1 (52), Grg5 (53) p300 (54), and Twists (55). Runx2 target genes include genes expressed by mature osteoblasts, such as osteocalcin, bone sialoprotein, osteopontin, and collagen 1(I) (22).
Little is known about how Osx regulates osteoblast differentiation and function. Expression of genes characteristic of mature osteoblasts is absent in cells surrounding chondrocytes in Osx null mice, and instead these cells express genes characteristic of chondrocytes. Thus, Osx may be important for directing precursor cells away from the chondrocyte lineage and toward the osteoblast lineage (44). Recently, an important role in osteoblast development for ?-catenin has become clear. ?-Catenin is the downstream mediator of canonical Wnt signaling that forms a transcription-regulating complex with TCF/LEF transcription factors. Inactivating mutations of LRP5 (low-density lipoprotein receptor-related protein 5), which encodes a Wnt coreceptor required for activation of canonical Wnt signaling, cause osteoporosis in humans (56) and in mice (57), whereas other mutations in LRP5 cause high bone mass (58, 59). Recent work in which ?-catenin is conditionally knocked out from cells at various stages of the osteoblast lineage, suggests that ?-catenin plays multiple critical roles in osteoblast differentiation (60, 61).
Alterations in functions of various other non-bone-specific transcription factors have been also demonstrated to affect osteoblastic differentiation and function. These include activator protein-1 and its related molecules (62), Dlx5 (63, 64), Msx1 (65), Msx2 (66, 67, 68), Twist (69), Atf4 (70), and nuclear steroid hormone receptors such as androgen receptors (71) and estrogen receptors (72).
Osteoclast Differentiation
Osteoclasts develop from monocytic precursors of the hematopoietic lineage. Analysis of a variety of mutant mice with osteopetrosis, caused by loss or impairment of osteoclast function, has provided valuable insights into the genetic basis for osteoclast differentiation. The Ets family transcription factor, PU.1, is responsible for the earliest established event in osteoclastogenesis. PU.1 null mice lack not only osteoclasts but also macrophages, while preserving the ability to produce early monocytic cells (73). After commitment to the osteoclast lineage, mononuclear cells respond to macrophage colony-stimulating factor (M-CSF), produced by nearby stromal cells, through activation of c-fms, the receptor for M-CSF; mice with inactivating mutations of M-CSF form macrophages normally, but lack osteoclasts (74). The other signaling system essential for osteoclast differentiation is triggered when RANKL [receptor activator of nuclear factor B (RANK) ligand], a member of the TNF family, activates its receptor RANK, a member of the TNF receptor family. Several transcription factors have been found crucial for osteoclast differentiation downstream of M-CSF/c-fms and RANKL/RANK signaling. Microphthalmia-associated transcription factors (MITF), transcription factor E (TFE3), TFEB, TFEC, and MITF, are essential for differentiation of mononuclear precursors into multinucleated osteoclasts (75). The phenotypic variance among different mutations in the MITF gene and analysis of compound mutants for mitf and tfe3 demonstrated that these family members have redundant roles (76). MITF directly regulates genes important for osteoclast function such as tartrate-resistant acid phosphatase (TRAP) (77, 78), cathepsin K (79), and osteoclast-associated receptor (OSCAR) (80) and also may regulate other transcription factors essential for osteoclastogenesis, such as PU.1 and c-Fos, by physical interaction (77, 81). The importance of NF-B was demonstrated by that the absence of multinucleated bone-resorbing cells in NF-B mutant mice (82). Development of macrophages is preserved in NF-B null mice, suggesting that NF-B functions later than PU.1 during osteoclast differentiation. In addition to NF-B, the RANK signaling pathway activates at least two other transcription factors essential for osteoclast differentiation: c-fos, a member of the activator protein-1 family of transcription factors, and NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1). The absence of c-fos causes severe osteopetrosis with lack of osteoclasts (83, 84). This defect is rescued by expression of a Fos-related molecule Fra1that lacks a transactivation domain; this suggests that Fos regulates osteoclast formation by interacting with other DNA binding molecules (85). C-fos is required for the initial induction of NFATc1 expression (86, 87). NFATc1, identified as a gene up-regulated in RANKL-stimulated bone marrow-derived monocyte/macrophage precursor cells (88, 89), plays a critical role in osteoclast differentiation (90). Unlike wild-type ES cells, ES cells missing the NFATc1 gene are not able to differentiate into osteoclasts in vitro. Furthermore, overexpression of a constitutively active form of NFATc1 in c-fos null cells restores expression of osteoclast-specific genes, demonstrating that NFATc1 is a critical transcriptional regulator downstream of c-fos during osteoclast differentiation (87).
We have briefly discussed transcription factors that have been shown through genetic studies to be important in bone development (see Fig. 1 for a summary of these transcription factors and critical signaling systems). Although animal models with genetic alterations demonstrate the physiological importance of genes, such models often give limited information about the primary targets of these transcription factors and subsequent cascades of gene expression. Therefore, the precise way that these transcription factors regulate bone cell development and function in vivo is limited and an important agenda for the future.
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Address all correspondence and requests for reprints to: Henry Kronenberg, Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114.
Abstract
Regulation of gene expression by transcription factors is one of the major mechanisms for controlling cellular functions. Recent advances in genetic manipulation of model animals has allowed the study of the roles of various genes and their products in physiological settings and has demonstrated the importance of specific transcription factors in bone development. Three lineages of bone cells, chondrocytes, osteoblasts, and osteoclasts, develop and differentiate according to their distinct developmental programs. These cells go through multiple differentiation stages, which are often regulated by specific transcription factors. In this minireview, we will discuss selected transcription factors that have been demonstrated to critically affect bone cell development. Further study of these molecules will lead to deeper understanding in mechanisms that govern development of bone.
BONE DEVELOPMENT IS achieved through the use of two distinct pathways, intramembranous and endochondral bone formation. In intramembranous bone formation, mesenchymal cells condense and directly differentiate into bone-forming osteoblasts. In contrast, in endochondral bone formation, mesenchymal cells condense and then become chondrocytes. This cartilage mold then directs the formation of osteoblasts, which form mature bone. This bone is continually remodeled by cycles of bone formation and resorption. Osteoclasts, of hematopoietic origin, are responsible for bone resorption. Thus, three distinct cell types (chondrocytes, osteoblasts, and osteoclasts) from two cell lineages direct the formation and remodeling of bone (1).
Endochondral Bone Formation and Chondrocyte Differentiation
Formation of the cartilage anlage starts with mesenchymal condensation (2). The molecular mechanism that regulates this process at the transcriptional level is not fully understood. However, because bone morphogenetic protein (BMP) signaling appears crucial for this process (3), Smads [a name derived from Sma and MADD (Mother against decapentaplegic)], transcription factors downstream of BMP signaling (4), likely play an important role. After the formation of mesenchymal condensations, cells start proliferating again and differentiate into chondrocytes (Fig. 1).
FIG. 1. Differentiation of bone cells of three lineages and its regulation by transcription factors. Transcription factors important for bone cell differentiation are indicated by bold black letters with arrows at major differentiation steps. Signaling molecules are indicated by blue letters. Osteoblasts and chondrocytes are derived from common mesenchymal precursors. During endochondral bone formation, mesenchymal cells condense and differentiate into chondrocytes. BMP signaling is likely required for these processes. Sox9 and its related molecules L-Sox5 and Sox6 play a pivotal role in commitment and maintenance of chondrocyte phenotypes. In the growth plate, chondrocytes proliferate and further differentiate into column-forming cells, then into postmitotic hypertrophic chondrocytes. Runx2 expression stimulates terminal differentiation. Runx2 and Osx are both required for osteoblast differentiation. Runx2 expression starts before that of Osx. Its activity is regulated by interaction with other transcription factors such as members of the Twist family. Osteoclasts differentiate from hematopoietic lineage cells through multiple steps. M-CSF and RANLK are essential external stimuli for osteoclastogenesis. PU.1, MITFs, Nf-B, Fos/Fra1, and NFATc1 are all required for differentiation of mature osteoclasts.
Sox9 (sex reversal Y-related high-mobility group box protein) and chondrocyte commitment
Analysis of genetically manipulated mice demonstrated that Sox transcription factors, members of the high mobility group superfamily, are necessary for conversion of condensed mesenchymal cells to chondrocytes. Studies of chimeric mice (5) and conditional knockouts (6) have shown that Sox9 is required for formation of normal mesenchymal condensations, for conversion of mesenchymal cells to chondrocytes, for proliferation of chondrocytes, and for suppression of premature conversion of these chondrocytes to hypertrophic chondrocytes. Sox9 is also required for the production of L-Sox5 and Sox6, two related Sox family members required for normal chondrocyte function (7). Sox9 is not only required for development of chondrocytes but also directly regulates expression of genes important for chondrocyte function, such as Col2a1 (8), Col11a2 (9), Agc1 (aggrecan) (10), and Mia (Cdrap) (11). The function of Sox9 may be modulated by phosphorylation by the PTH-related peptide signaling pathway (12). It is, therefore, possible that Sox9 mediates part of PTH-related peptide action to regulate hypertrophic differentiation.
Expression of Sox9 begins in the mesenchymal condensation. BMPs can induce SOX9 expression, and noggin, a BMP antagonist, blocks expression of SOX9 in mesenchymal condensations (13). Cytokines such as IL-1 and TNF-, and nuclear factor B (NF-B), the transcription factor that mediates many actions of these cytokines, suppress Sox9 expression, partly through induction of posttranscriptional mRNA degradation (14, 15); this suppression of SOX9 probably contributes to chondrocyte loss in inflammatory arthritis. How specific transcription factors regulate Sox9 gene transcription is a central question, although it appears complicated; genetic analysis of human and mouse Sox9 mutations demonstrated that cis elements quite distant from the transcription start site of the Sox9 gene are critical for Sox9 transcription in cartilage (16).
Interestingly, overexpression of Sox9 in the cartilage causes a decrease in chondrocyte proliferation and a delay in bone development. This decrease in proliferation may result from binding of Sox9 to ?-catenin, the essential component of the canonical Wnt (a name that combines wingless and int) signaling pathway (17). Some Wnt members are expressed in cartilage, and effects of Wnt signaling in cartilage have been demonstrated (18, 19, 20, 21). Along with the observation that overactivation or deletion of ?-catenin in chondrocytes resulted in severe skeletal dysplasias, these findings suggest that the transcription complex, lymphoid enhancer-binding factor (LEF)/T-cell factor (TCF)/?-catenin, regulates some aspects of cartilage development (17).
Runx2 and chondrocyte hypertrophy
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 because chondrocytes stop dividing and become hypertrophic. Runx transcription factors are members of the runt family, named for the DNA binding domain, conserved across species from Drosophila to humans (22). Mice missing Runx2 show a defect in chondrocyte maturation, with lack of hypertrophic chondrocytes in many bones (23), and mice missing both Runx2 and Runx3 completely lack chondrocytes (24). Furthermore, overexpression of Runx2 hastens hypertrophy and even converts chondrocytes in the trachea, which normally never hypertrophy, into hypertrophic chondrocytes and then bone (25, 26).
The stages of chondrocyte differentiation are regulated by a complex series of signaling molecules and transcription factors in addition to Sox9 and Runx2/3. Differentiation of early periarticular chondrocytes into flat, columnar proliferating chondrocytes appears to be regulated by multiple signaling molecules such as Indian hedgehog (Ihh) and Wnts (19, 27). It is, therefore, likely that some of the downstream transcription factors triggered by Ihh and wnts, Gli family members, and the ?-catenin/TCF/LEF complex, play a role in this step. Chondrocyte differentiation is accompanied by changes in proliferation. Alterations in fibroblast growth factor (FGF) signaling (28, 29), BMP signaling (30), and Ihh signaling (31, 32, 33) participate in regulating chondrocyte proliferation. The suppressive effect of fibroblast growth factor signaling on proliferation is regulated by signal transducer and activator of transcription-1 at the transcriptional level (34). Overexpression of Ihh is associated with an increase in cyclin D1 expression (33), which ultimately stimulates cell cycle progression by activating E2F transcription factors (35). BMP signaling, therefore, involving Smad 1, 5, and 8 transcription factors, generally stimulates hypertrophic differentiation (30, 36, 37, 38, 39). Misexpression of a homeobox-containing transcription factor, Dlx5, which is normally expressed in the prehypertrophic region of the growth plate, stimulates hypertrophic differentiation in developing chicken limbs (40). Considering that Dlx5 is downstream of BMP signaling in several other cell types, Dlx5 and its related molecule, Dlx6 (41), may mediate BMP action regulating hypertrophic differentiation. These animal models with Dlx modulation, however, need cautious interpretation because neither misexpression nor deletion of the genes is chondrocyte specific.
Osteoblast Differentiation
Two transcription factors, Runx2 containing a runt domain (42, 43) and Osterix (Osx; SP7) with a zinc-finger motif (44) are absolutely required for osteoblast differentiation during both intramembranous and endochondral bone formation. Analysis of Osx null mice shows that Osx is genetically downstream of Runx2. Runx2 is expressed in the lateral mesoderm, mesenchymal condensations, and chondrocytes in addition to osteoblasts. Both overexpression of Runx2 and expression of a dominant-negative form of Runx2 in osteoblasts impairs bone formation, suggesting that regulation of different stages of osteoblast differentiation by Runx2 is complex (45, 46). Runx2 is regulated by phosphorylation and also interacts with other transcription factors, such as Smads 1 and 5 (47, 48), Smad 3 (49), signal transducer and activator of transcription-1 (50), Menin (51), Hey1 (52), Grg5 (53) p300 (54), and Twists (55). Runx2 target genes include genes expressed by mature osteoblasts, such as osteocalcin, bone sialoprotein, osteopontin, and collagen 1(I) (22).
Little is known about how Osx regulates osteoblast differentiation and function. Expression of genes characteristic of mature osteoblasts is absent in cells surrounding chondrocytes in Osx null mice, and instead these cells express genes characteristic of chondrocytes. Thus, Osx may be important for directing precursor cells away from the chondrocyte lineage and toward the osteoblast lineage (44). Recently, an important role in osteoblast development for ?-catenin has become clear. ?-Catenin is the downstream mediator of canonical Wnt signaling that forms a transcription-regulating complex with TCF/LEF transcription factors. Inactivating mutations of LRP5 (low-density lipoprotein receptor-related protein 5), which encodes a Wnt coreceptor required for activation of canonical Wnt signaling, cause osteoporosis in humans (56) and in mice (57), whereas other mutations in LRP5 cause high bone mass (58, 59). Recent work in which ?-catenin is conditionally knocked out from cells at various stages of the osteoblast lineage, suggests that ?-catenin plays multiple critical roles in osteoblast differentiation (60, 61).
Alterations in functions of various other non-bone-specific transcription factors have been also demonstrated to affect osteoblastic differentiation and function. These include activator protein-1 and its related molecules (62), Dlx5 (63, 64), Msx1 (65), Msx2 (66, 67, 68), Twist (69), Atf4 (70), and nuclear steroid hormone receptors such as androgen receptors (71) and estrogen receptors (72).
Osteoclast Differentiation
Osteoclasts develop from monocytic precursors of the hematopoietic lineage. Analysis of a variety of mutant mice with osteopetrosis, caused by loss or impairment of osteoclast function, has provided valuable insights into the genetic basis for osteoclast differentiation. The Ets family transcription factor, PU.1, is responsible for the earliest established event in osteoclastogenesis. PU.1 null mice lack not only osteoclasts but also macrophages, while preserving the ability to produce early monocytic cells (73). After commitment to the osteoclast lineage, mononuclear cells respond to macrophage colony-stimulating factor (M-CSF), produced by nearby stromal cells, through activation of c-fms, the receptor for M-CSF; mice with inactivating mutations of M-CSF form macrophages normally, but lack osteoclasts (74). The other signaling system essential for osteoclast differentiation is triggered when RANKL [receptor activator of nuclear factor B (RANK) ligand], a member of the TNF family, activates its receptor RANK, a member of the TNF receptor family. Several transcription factors have been found crucial for osteoclast differentiation downstream of M-CSF/c-fms and RANKL/RANK signaling. Microphthalmia-associated transcription factors (MITF), transcription factor E (TFE3), TFEB, TFEC, and MITF, are essential for differentiation of mononuclear precursors into multinucleated osteoclasts (75). The phenotypic variance among different mutations in the MITF gene and analysis of compound mutants for mitf and tfe3 demonstrated that these family members have redundant roles (76). MITF directly regulates genes important for osteoclast function such as tartrate-resistant acid phosphatase (TRAP) (77, 78), cathepsin K (79), and osteoclast-associated receptor (OSCAR) (80) and also may regulate other transcription factors essential for osteoclastogenesis, such as PU.1 and c-Fos, by physical interaction (77, 81). The importance of NF-B was demonstrated by that the absence of multinucleated bone-resorbing cells in NF-B mutant mice (82). Development of macrophages is preserved in NF-B null mice, suggesting that NF-B functions later than PU.1 during osteoclast differentiation. In addition to NF-B, the RANK signaling pathway activates at least two other transcription factors essential for osteoclast differentiation: c-fos, a member of the activator protein-1 family of transcription factors, and NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1). The absence of c-fos causes severe osteopetrosis with lack of osteoclasts (83, 84). This defect is rescued by expression of a Fos-related molecule Fra1that lacks a transactivation domain; this suggests that Fos regulates osteoclast formation by interacting with other DNA binding molecules (85). C-fos is required for the initial induction of NFATc1 expression (86, 87). NFATc1, identified as a gene up-regulated in RANKL-stimulated bone marrow-derived monocyte/macrophage precursor cells (88, 89), plays a critical role in osteoclast differentiation (90). Unlike wild-type ES cells, ES cells missing the NFATc1 gene are not able to differentiate into osteoclasts in vitro. Furthermore, overexpression of a constitutively active form of NFATc1 in c-fos null cells restores expression of osteoclast-specific genes, demonstrating that NFATc1 is a critical transcriptional regulator downstream of c-fos during osteoclast differentiation (87).
We have briefly discussed transcription factors that have been shown through genetic studies to be important in bone development (see Fig. 1 for a summary of these transcription factors and critical signaling systems). Although animal models with genetic alterations demonstrate the physiological importance of genes, such models often give limited information about the primary targets of these transcription factors and subsequent cascades of gene expression. Therefore, the precise way that these transcription factors regulate bone cell development and function in vivo is limited and an important agenda for the future.
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