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Chapter 9
Osteoblast Biology
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FIGURE 9.1 Lineage allocation of stem cells. A. Representation of stem cell renewal and maturation to the
mesenchymal stromal cell with limited pluripotency. The transcription factors proven through genetic studies to
function as master regulatory genes required for the indicated phenotypes are shown. B. Transcriptional
regulation of lineage determination and the role of Runx2 expressed in the undifferentiated mesenchymal cell is
indicated. Runx2 will inhibit other cell phenotypes including the myoblasts (not shown). For cells to enter the
chondrogenic lineage, Runx2 must be downregulated, and several transcription factors including Sox9 and Twist
are negative regulators of Runx2. The downregulation of Nkx3.2 permits reactivation of Runx2 expression in the
hypertrophic chondrocyte. Adipogenesis and osteoblastogenesis can be regulated by expression of either Runx2
or PPARγ. A sampling of Runx2 target genes that reflect the different cell phenotypes and Runx2 functions for
bone formation are shown.
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FIGURE 9.2 Osteoblast lineage cells. A. Stages of osteoblast maturation are visualized on the surface of this
bone trabeculae, Goldner trichome stain. B. Mouse cortical bone from a transgenic mouse expressing green
fluorescent protein under control of the osteocalcin promoter is shown to illustrate that this bone-specific marker
is expressed in osteoblasts and osteocytes. C. An osteon of human bone shows circumferential layers of cells
and tissue around the Haversian Canal (HC). The osteocyte cell body (indicated by OC) in lacunae with dendritic
process in canaliculi (CAN) are visualized. D. Low magnification of electron micrograph of a demineralized
osteon showing the lamellar organization of the matrix (L1, L2, L3 layers) with active osteoblasts on the surface.
E. Cuboidal osteoblasts (arrows) staining positive for osteocalcin and lining the osteoid (triangle). Source: Boden
et al. (1998) [73].
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FIGURE 9.3 Stages of osteoblast differentiation in vitro. A. Histologic staining by Toluidine blue (left panel),
alkaline phosphatase (middle panel), and von Kossa silver stain (right panel) to reflect the major stages of
osteoblast maturation. B shows expression of marker genes reaching peak expression that is characteristic of
each stage. C. Transcription factor expression is represented. Several factors are rapidly induced in pluripotent
stem cells in response to osteogenic bone morphogenetic proteins (BMPs) (see text). Runx2 continuously
increases during osteoblast differentiation. Dlx5, Osterix, and ATF4 are functionally linked to the late
mineralization stage [353,354,363,540]. D. Diagrams binding homeodomain proteins to gene promoters during
differentiation [362,363]. The association and dissociation of these factors at the TAAT core motif in genes form a
regulatory network to support transcription. ECM: extracellular matrix.
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FIGURE 9.4 Immunostaining for osteopontin. Osteopontin is detected at the bone surface, between bone
surface and osteoclast, and around the osteocytes as indicated by the arrows. Osteoclast is indicated by the
arrowhead [73].
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FIGURE 9.5 Control of cell cycle progression in bone cells. Progression through the cell cycle is controlled by
formation of cyclin and cyclin-dependent kinase (cdk) complexes at each stage (M, G1, S, and G2). Activities
associated with each stage are indicated. Entry into/from G0 and exit from the cell cycle is controlled by growthregulatory factors (e.g., cytokines, growth factors, cell adhesion, and/or cell–cell contact) that determine selfrenewal of stem cells and expansion of precommitted progenitor cells. The cell cycle is regulated by several
critical cell cycle checkpoints (indicated by checkmarks), at which competency for cell cycle progression is
monitored. The biochemical parameters associated with each cell cycle checkpoint are indicated. Options for
defaulting to apoptosis during G1 and G2 are evaluated by surveillance mechanisms that assess fidelity of
structural and regulatory parameters of cell cycle control. Apoptosis also occurs in mature differentiated bone
cells.
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FIGURE 9.6 Nuclear architecture contributes to bone-specific gene regulation. A. Levels of chromatin
organization. Chromatin organization and the nucleosome of core histone protein for binding deoxyribonucleic
acid (DNA) (left panel). Post-translational modifications of histone proteins regulate active (open) chromatin and
inactive (condensed) chromatin. Chromatin loop domains (10–100 kb) are tethered to components of the nuclear
matrix through MAR (matrix attachment regions) sequences. An individual gene with a positioned nucleosome is
illustrated within the loop (right panel). B. Electron micrograph of the filamentous structure of the nuclear matrix
scaffold [500]. With examples of organization of functional activities in domains associated with the nuclear
matrix scaffold [541]. Antibodies to markers of the indicated functional domains reveal the organization of
structures and transcriptional foci. C. The Runx2-Smad interaction occurs by recruitment of TGF-β and BMPinduced Smad proteins to Runx2 domains in the nuclear matrix compartment. Shown is the interaction of Runx2
and the BMP2-induced Smad1 in situ in HeLa cells transfected with XPRESS tag Runx2 and flag tagged Smad
[542].
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FIGURE 9.7 Regulatory elements andchromatin organization of the osteocalcin gene promoter. A. Illustrates the
transcriptionally active osteocalcin gene indicated by strong DNase hypersensitivity (DHS) between the proximal
and distal domains separated by a positioned nucleosome within the 1.1-kb promoter. A single tissue-specific
homeodomain (HD) element in the designated conserved “osteocalcin” (OC) box contributes to osteoblast stagespecific expression. The OC gene is under repression by Msx2 during proliferation, then activated by Dlx3 and
Dlx5 during postproliferative differentiation. The multiple Runx sites regulate tissue-specific expression and
physiological responses. B. Three-dimensional model of OC promoter structure is based on experimental
evidences including (i) the positioned nucleosome in the transcribed gene shortening the distances between
proximal and distal domains for protein–protein interactions, (ii) direct physical interactions between the vitamin
D receptor (VDR) and Runx2, as well as between the VDR and TFIIB, and (iii) mutation of the Runx sites, which
decreases DNA accessibility to regulatory factors. The association of Runx with the nuclear matrix scaffold
supports gene promoter conformation and regulatory element cross-talk, facilitating interaction between the
proximal RNA polymerase complex interactions with the VDR complex. This mechanism allows for physiologic
upregulation of osteocalcin by vitamin D coordinated with basal transcriptional levels.
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FIGURE 9.8 Domain organization of Runx2 and interacting proteins. The organization of Runx2 regulatory
elements showing a nuclear localization signal (NLS) contiguous to the runt homology DNA binding domain
(RHD) and a second intranuclear trafficking signal designated the nuclear matrix targeting signal (NMTS). A
Smad-interacting motif of three amino acids (HTY) overlaps the NMTS [543] in the C-terminus. The C-terminal
Groucho/TLE interacting protein is also nuclear matrix associated with its own distinct targeting signal [544].
Illustrated are examples of proteins that form complexes with Runx2 altering Runx2 transcriptional activity,
providing mechanisms for positive and negative Runx2-mediated gene expression as a cell progresses through
stages of differentiation or in response to physiologic signals that affect bone metabolism (see text). PST,
proline-serine-threonine.
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FIGURE 9.10 Regulation of bone formation and osteoblast differentiation by selected micro-RNAs.
The illustration of osteoblast lineage cells is presented in the context of three different functional
activities of micro-RNAs in the skeleton (gray filled boxes 1, 2, 3). One micro-RNA can regulate
multiple stages by targeting different proteins, e.g., miR-29. The downregulation of micro-RNAs
that target osteogenic factors (e.g., Runx2, Smad) in mesenchymal stromal cells (MSCs)
facilitates induction of osteoblast differentiation. The presence of any one of the micro-RNAs
inhibiting Runx2 in nonosseous cells is a mechanism for controlling lineage fate by micro-RNAs.
Several of the micro-RNAs that are upregulated in mature osteoblasts/osteocytes contribute to
regulation of bone mass as revealed by loss-of-function mutation of micro-RNAs in osteoblasts
that results in a high bone mass phenotype.
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FIGURE 9.9 Mechanism by which Runx2 supports lineage commitment by association with mitotic
chromosomes. Saos cells (human osteoblastic osteosarcoma) were stained with either endogenous proteins
α-tubulin (red), Runx2 (green), or for DNA with DAPI (blue). A. Resting cell in interphase; B. Cell in mitosis; C.
Runx2 foci on chromosomes [545]; and D. Equal distribution of Runx2 in the two daughter cells [520]. This
association of Runx2 may function in bookmarking target genes for postmitotic osteogenic lineage determination.
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