1. Chapter 9 Chapter 9 Osteoblast Biology Copyright © 2013 Elsevier Inc. All rights reserved.

2. 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. 2

3. Copyright © 2013 Elsevier Inc. All rights reserved. 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]. 3

4. Copyright © 2013 Elsevier Inc. All rights reserved. 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. 4

5. Copyright © 2013 Elsevier Inc. All rights reserved. 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]. 5

6. Copyright © 2013 Elsevier Inc. All rights reserved. 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, G 1, S, and G 2 ). Activities associated with each stage are indicated. Entry into/from G 0 and exit from the cell cycle is controlled by growth- regulatory factors (e.g., cytokines, growth factors, cell adhesion, and/or cell–cell contact) that determine self- renewal 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 G 1 and G 2 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. 6

7. Copyright © 2013 Elsevier Inc. All rights reserved. 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 BMP- induced 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]. 7

8. Copyright © 2013 Elsevier Inc. All rights reserved. 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 stage- specific 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. 8

9. Copyright © 2013 Elsevier Inc. All rights reserved. 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. 9

10. Copyright © 2013 Elsevier Inc. All rights reserved. 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. 10

11. Copyright © 2013 Elsevier Inc. All rights reserved. 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. 11