The differentiation of osteoblasts from mesenchymal progenitors, which also originate chondrocytes, myoblasts, from adipocytes, and tendon cells, requires the activity of specific transcription factors that are expressed at distinct time points during the differentiation process, thereby defining various developmental stages of the osteoblast lineage.

Runt domain–containing transcription factor (Runx2 ) is a master switch for osteoblast differentiation. Levels of Runx2 gradually increase in subsequent stages of osteoblast differentiation, with maximum expression observed in the mature osteoblasts. Homozygous deletion of Runx2 in mice resulted in a complete lack of osteoblasts [7]. Runx2 is both necessary and sufficient for mesenchymal cell differentiation toward the osteoblast lineage [8]. It was demonstrated that Runx2 controls bone lineage cells by binding to the Runx regulatory element in promoters of osteoblastogenic genes. Runx2 target genes include genes expressed by both immature and differentiated osteoblasts. Such as TGF-β receptor, ALP, collagen type I alpha 1 and alpha 2 chain, OPN, OSTC, Vitamin D receptor, BSP, ON, and collagenase [9]. Thus, Runx2 is necessary for both osteoblast differentiation and function.

Osterix is another DNA-binding transcription factor that is absolutely required for osteoblast differentiation. Osterix/SP7 is a member of the zinc-finger-containing SP family and is abundantly expressed throughout osteoblast differentiation. Genetic inactivation of Osterix in mice results in the absence of mineralized bone matrix, defective osteoblasts, and perinatal lethality [10]. Similar to Runx2, forced expression of Osterix in non-bone cells, promotes expression of both early and late marker genes of osteoblasts. However, molecular and genetic studies revealed that Runx2 is expressed in mesenchymal tissues of Osterix null mice [10]. Thus, Osterix acts downstream of Runx2 in the transcriptional cascade of osteoblast differentiation. Consistently, Osterix expression is positively regulated by direct binding of Runx2 to a responsive element in the promoter of the Osterix gene.

Activating Transcription Factor 4 (ATF4 ), a member of the basic Leu zipper family of transcription factors, has important roles in the more mature osteoblast lineage cells. Misregulation of ATF4 activity has been linked with the skeletal abnormalities seen in human patients with Coffin–Lowry syndrome and neurofibromatosis type I [11]. ATF4 may function in osteoblast lineage cells through two distinct mechanisms. First, it directly regulates the expression of OSTC and RANKL [11]. Second, ATF4 promotes efficient amino acid import to ensure proper protein synthesis by osteoblasts [11].

Activator Protein 1 transcription factor family (AP1 ) is composed of heterodimers of Fos-related factors (cFos, Fra1, Fra2, and FosB) and Jun proteins (cJun, JunB and JunD). Multiple Fos and Jun proteins are highly expressed in proliferating osteoprogenitors. Their expression decreases during differentiation, except for Fra2 and JunD that are the primary AP1 components present in mature osteoblasts [12]. Some observations indicated that c-Fos [12], Fra1, ΔFosB (an alternative splice variant of FosB) [12], and JunB [13] proteins promote bone formation. In contrast, JunD promotes a decrease in bone mass repressing other AP1 protein expressions in osteoblasts [14]. A number of direct targets of AP1 in osteoblasts have been identified, and include the OSTC, collagenase-3, BSP, and ALP promoters [12].

Skeletal cells produce numerous growth factors and cytokines that signal in both autocrine and paracrine way to control cell proliferation, differentiation, and survival. These secreted factors and signaling pathways either promote or suppress the expression of transcription factors essential for osteoblast differentiation.

Transforming Growth Factor–β (TGF-β) is one of the most abundant cytokines in bone matrix and plays a major role in development and maintenance of the skeleton, affecting both cartilage and bone metabolism [15]. By binding to its specific receptors, TGF-β induces activation of Smad-2 and Smad-3 intracellular proteins that transduce extracellular signals from TGF-β to the nucleus, regulating osteoblast gene expression [16]. TGF-β is important in the maintenance and expansion of the mesenchymal stem/progenitor cells. TGF-β signaling also promotes osteoprogenitor proliferation, early differentiation and commitment to the osteoblastic lineage through the Smad2/3 pathways, and the cooperation between TGF-β and PTH, Wnt, BMP, as well as FGF signaling.

Bone morphogenetic proteins (BMPs) belonging to the TGF-β superfamily were originally identified as the active components in bone extracts capable of inducing ectopic bone formation. BMPs are expressed in skeletal tissue and are required for skeletal development and maintenance of adult bone homeostasis and play an important role in fracture healing [17]. Genetic studies have demonstrated an important role for BMP2 and BMP4 in promoting osteoblast differentiation and function [18]. Recent studies have also shown that BMP3 inhibits the signal transduced by BMP2 or BMP4 [19], working as a negative regulator of osteoblast differentiation.

The Wingless (Wnt ) family of glycoproteins has recently emerged as central regulators of bone mass. Upon engaging various membrane receptors, Wnt ligands activate numerous intracellular pathways that are either dependent or independent on β-catenin. In the β-catenin-dependent signaling, Wnt binds to Frizzled receptors and their co-receptors low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) to stabilize cytosolic β-catenin that enters the nucleus and stimulates the transcription of Wnt target genes such as Runx2 and Osterix [20]. Thus, activation of the canonical pathway promotes the differentiation of osteoblast progenitor cells into mature osteoblasts. Wnt signaling is tightly regulated by a delicate balance of extracellular agonists and antagonists. There are different antagonists, such as soluble frizzled-related proteins (sFRPs), which inhibit Wnt signaling by binding to and sequestering Wnt ligands and others belonging to the Dickkopf (DKK), family members or the SOST gene product (sclerostin) that bind to and sequester the Wnt co-receptors LRP5/6 [21]. In human, receptor mutations that render Wnt signal constitutively active result in a generalized increase in bone mass [22]. Loss-of-function mutations in the gene encoding the Wnt co-receptor LRP5 cause osteoporosis-pseudoglioma syndrome [23], a form of juvenile-onset osteoporosis. Conversely, mutations in LRP5 that inhibit the interaction between the co-receptor and DKK1 or sclerostin cause high-bone mass syndrome [24, 25]. In addition, loss-of-function or loss-of-expression mutations in SOST result in the bone-thickening diseases, sclerosteosis or Van Buchem disease, respectively [26, 27]. β-catenin-independent Wnt signaling has also been implicated in promoting osteoblast differentiation [28]. In particular, Wnt5A is thought to promote osteoblast differentiation by inhibiting the activation of adipogenic genes [29]. Thus, both β-catenin-dependent and β-catenin-independent Wnt signaling are able to control differentiation of osteoblast progenitors into mature osteoblasts.

Fibroblast Growth Factors (FGFs ) are a large family of proteins (23 different ligands) that transduce their signal through one of the four FGF receptors (FGFR). FGFs initiate condensation of the mesenchyme and proliferation of progenitor cells. In particular, FGF2 is important for preosteoblast proliferation and maturation [30], while FGF18 is essential in mature osteoblast formation [31].

Osteocytes account for about 95 % of cells in the mature bone tissue. They are entombed individually in lacunae of the mineralized matrix and have cytoplasmic processes that radiate from the cell body and give osteocytes the aspect of neuronal cells. The processes run along canaliculi and are linked by gap junctions with the processes of neighboring osteocytes, as well as with cellular elements of the bone marrow, endothelial cells of the bone marrow, and bone surface cells including the lining cells. In contrast to osteoclasts and osteoblasts, which are relatively short-lived and transiently present only on a small fraction of the bone surface, osteocytes are long-lived cells and are deployed throughout the skeleton.

Nowadays, osteocytes are considered the choreographers of the remodeling process. They are able to direct the homing of osteoclasts to the site that is in need of remodeling [32] and to control and modify mineralization of the matrix produced by osteoblasts [33]. Moreover, they produce factors that influence mineral homeostasis and also mediate the homeostatic adaptation of bone to mechanical loading. Osteocytes are considered mechanosensory cells because of their peculiar location in bone and complex dendritic network. It was demonstrated that osteocyte may sense load through the cell body, the dendritic processes and the cilia [34]. Moreover, it was demonstrated that 20 % of the osteocytes are active in bone formation [35] and that could surprisingly directly remove minerals from their lacunae and canaliculi, playing a role in mineral homeostasis during a calcium-demanding condition such as lactation [36]. Literature data also demonstrate the ability of osteocytes to negatively regulate osteoblast formation and activity through the secretion of sclerostin, a well known Wnt inhibitor [37]. Recently, the ability of osteocytes to influence osteoclast formation through the secretion of the osteoclastogenic cytokine RANKL was also showed [38].

Osteoclasts are terminally differentiated multinucleated cells that are the principal resorptive cells of bone, playing a central role in the formation of the skeleton and regulation of its mass [45, 46]. The osteoclasts are usually found in contact with a calcified bone surface and within a lacuna (Howship’s lacunae) that is the result of its own resorptive activity. The zone of contact with the bone is characterized by the presence of a ruffled border, which consists of complex folds of plasma membrane juxtaposed to bone and surrounded by the actin ring or sealing zone. The ruffled border and the sealing zone appear only in osteoclasts adherent to the mineralized matrix and in a resorptive state. The sealing zone is formed by a ring of focal points of adhesion (podosomes) with a core of actin and several cytoskeletal and regulatory proteins around it that attach the cell to the bone surface, thus sealing off the subosteoclastic bone-resorbing compartment. The attachment of the cell to the matrix is performed via integrin receptors, which bind to specific RGD (Arginine-Glycine-Aspartate) sequences found in matrix proteins, and the αvβ3 is the integrin predominantly expressed by osteoclasts. The plasma membrane in the ruffled border area contains proteins that are also found at the limiting membrane of lysosomes and a specific type of electrogenic vacuolar proton ATPase involved in acidification. The basolateral plasma membrane of the osteoclast is specifically enriched with Na+, K+-ATPase, HCO₃ ⁻/Cl⁻, and Na+/H+ exchangers [47]. Lysosomal enzymes such as tartrate-resistant acid phosphatase and cathepsin K are secreted, via the ruffled border, into the extracellular bone-resorbing compartment and are involved in bone matrix digestion.