The progression of osteoblast maturation requires a sequential activation and suppression of genes that encode the phenotypic and functional proteins of the osteoblast populations. Signaling proteins, transcription factors and regulatory proteins support the temporal expression of genes that characterize stages of osteoblast differentiation and extracellular matrix synthesis and mineralization (bone formation) (Figure 2).
Osteoprogenitor cells arise from multipotential mesenchymal stem cells that give rise to a number of cell lineages including those for osteoblasts (OB) (2). The differentiation towards the osteoblastic lineage is controlled by genes related to the Hedgehog family among which Indian hedgehog (Ihh) and Sonic hedgehog (Shh) are implicated (3, 4). The transcription factors such as Cbfa1 (core-binding protein a-1, also known as Runx2) and osterix (5) are crucial for osteoblastic differentiation.
Signaling molecules such as -catenin activated by Wnt proteins (6-8) (see below) and Smad proteins activated by the bone morphogenic proteins (BMPs) are also powerful regulators of osteoblastic differentiation from the mesenchymal stem cells (Figure 2) (4).
Differentiation towards the osteoblastic versus adipocytic lineage strongly depends on specific transcription factors such a Cbfa1 for the osteoblastic lineage (9-11) and PPAR (peroxisome proliferator-activated receptor gamma) for the adipocytic lineage (12). In fact, when PPAR is more expressed than Cbfa1, differentiation towards the adipocytic lineage is more important and thus induces a decreased number of osteoblasts (‘Fat gain is bone loss’). This correlates with the fact that with ageing bone marrow adipogenesis is increased (13) and moreover, PPAR activators such as glitazones decrease bone mass and are associated with an increased risk of fractures (14-17). Another transcription factor implicated in bone formation is Sox4.
In fact, Sox4 knockout mice are lethal but hererozygote mice have an intrinsic osteoblast defect that produces cortical and trabecular osteopenia. Proliferation,
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differentiation and mineralization of cultured Sox4+/- osteoblasts is dramatically impaired.
Among the local signaling factors implicated in the remodeling process are Wnt ligands. Their role was inferred after the identification of mutations in the Wnt co-receptor low-density lipoprotein co-receptor-related protein 5 (Lrp5) in patients with heritable skeletal diseases. ‘Loss-of-function’ mutations in Lrp5 were found to cause the Osteopetrosis-Pseudoglioma syndrome (OPPG), an autosomal recessive disorder characterized by extremely low BMD and skeletal fragility (18). Missense mutation in Lrp5 that are thought to create a ‘gain of function’ cause autosomal dominant high bone mass phenotypes in which BMDs are well above the population mean (19, 20).
Lrp5, a single-transmembrane-domain receptor, interacts with the frizzled receptor complex to inhibit the phosphorylation of -catenin by glycogen synthetase kinase-3 (GSK-3). As phosphorylated -catenin is more susceptible to ubiquitin-mediated degradation, by inhibiting GSK-3 activity, Lrp5 allows accumulation of -catenin and its nuclear ingress. Activated -catenin then interacts cooperatively with Tcf/Lef transcription factors to stimulate osteoblast differentiation. Mutations in Wnt members, ablation of the Wnt co-receptor Lrp5 or Lrp6, or use of Wnt inhibitors such as sclerostin, soluble frizzled-related proteins, Wnt inhibitors factors and the Dickkoff family members Dkk1 and Dkk2, reduce osteoblastogenesis and bone formation (21-24).
Once the osteoblastogenic cells are committed towards the osteoblastic lineage, they will temporally express proteins involved in extracellular matrix biosynthesis and mineralization. The extracellular matrix is extremely abundant, composed of collagen fibers (type I, 90% of the total protein) usually oriented in a preferential direction, and noncollagenous proteins (ground substance) such as glycoproteins, proteoglycans, osteopontin (OPN), bone sialoprotein (BSP) and bone gla-protein (BGP) also known as osteocalcin. The expression of these proteins provide a panel of osteoblast phenotypic markers that reflect stages of osteoblastic differentiation (Figure 1). Based on bone nodule formation in vitro, the process has been subdivided into three stages: 1) proliferation, 2) extracellular matrix development and
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maturation, and 3) mineralization, with characteristic changes in gene expression at each stage. Thus, the proliferation phase is associated with the expression of early genes such as c-fos and histone H4 (25, 26). Then, expression of the osteoblast-associated genes collagen type I (COLL I), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (BGP), bone sialoprotein (BSN), and parathyroid hormone receptor (PTH1R) is asynchronously upregulated, acquired, and/or lost as the osteoprogenitor cell differentiate and extracellular matrix matures and mineralizes (Figure 2) (27). In general, ALP increases then decreases when mineralization is well progressed. ALP catalyses the hydrolysis of phosphate esters at the osteoblast cell surface to provide a high phosphate concentration for bone mineralization process.
OPN peaks twice, during proliferation and then again later but before certain other matrix proteins including BSP and BGP. OPN as well as BSP are proteins that link bone cells and the hydroxyapatite of mineralized matrix. BSP is transiently expressed very early and then upregulated again in differentiated osteoblasts. Osteocalcin (BGP) appears approximately concomitantly with mineralization (27). BGP is thought to play a role in mineralization and calcium homeostasis. It has been postulated that it may also function as a negative regulator of bone formation (28).
Moreover, very recently osteoblast-secreted osteocalcin has been found to exert an endocrine regulation of sugar homeostasis (29). In fact, mice deficient in the osteoblastic-specific protein kinase OST-PTP have hyperinsulinemia, hypoglycemia and increased sensitivity to insulin, demonstrating that bone exerts humoral control over both pancreatic -cells and insulin target tissues. Osteocalcin knockout mice have an opposite phenotype, and removing one osteocalcin allele corrects the OST-PTP phenotype. Moreover, administraion of uncarboxylated osteocalcin improves glucose tolerance in vivo.
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Cbfa1
Osteoprogenitor Preosteoblast Mature osteoblast
Figure 2. Growth and differentiation of osteoblasts. A. Temporal expression of osteoblast genes during the stages of proliferation, matrix maturation and mineralization. B. The reciprocal and functionally coupled relationship between cell growth and differentiation-related gene expression is illustrated by arrows. Broken vertical lines between the three principal stages indicate transition points requiring downregulation of cell growth and extracellular matrix signaling events for the progression of differentiation. (COLL I: collagen type I; OPN: osteopontin; ALP:
alkaline phosphatase; BSP: bone sialoprotein; BGP: osteoclacin; OPN: osteopontin).
In summary, osteoblasts are key cells for bone formation during skeletal growth, renewal of bone matrix and bone repair. Their functions are 1) to synthesize collagen I and non collagenous proteins (ALP, BSP, OPN, OC, OPN) of the bone extracellular matrix; 2) direct the arrangement of the extracellular matrix fibrils; 3) contribute to the mineralization of bone matrix due to ALP that increases levels of phosphate (necessary for mineralization) by degrading inorganic pyrophosphates; 4) synthesize growth factors for the autocrine/paracrine regulation of bone formation, such as insulin growth factor (IGF) I and II, transforming growth factor (TGF-) and bone morphogenetic proteins (BMPs) (30); and 5) synthesize cytokines and other
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molecules for the coupling of osteoblasts to osteoclasts in order to regulate bone remodelling.