Osteomimicry: old concepts and new findings

Osteoblast differentiation

Osteoblasts are typically known as the bone-building cells of the bone tissue, representing approximately 4%–6% of total bone cells. When looking at a histological bone section, osteoblasts usually appear arranged in a row, lining the bone trabecular surfaces and having a cuboidal, epithelial-like morphology ( Fig. 11.1 ). Indeed, osteoblasts are mesenchymal cells originating from a pluripotent mesenchymal stem cell (MSC), which they share with other cell types, such as fibroblasts, adipocytes, chondrocytes, myoblasts, and tenocytes [ ].

One of the earliest pivotal events for osteoblast differentiation is the transit from an MSC to an osteoprogenitor cell, owing to the “switching on” of a transcription factor that is essential for osteoblast lineage commitment: Runt-related transcription factor 2 (Runx2), alias core-binding factor 1/osteoblast-specific factor 2 (Cbfa1/Osf2) [ ], whose absence is lethal in mouse models. Mice carrying a homozygous mutation in Runx2 die just after birth due to breathing problems. Moreover, they present with the lack of both endochondral and intramembranous ossification, due to failure in osteoblast maturation [ ], while heterozygous mutants recapitulate the skeletal defects observed in the autosomal dominant genetic disease cleidocranial dysplasia (CCD) [ ]. Runx2 promotes the expression of early osteogenic markers, such as alkaline phosphatase (Alp) and collagen I.

Moving on to the differentiation process, another transcription factor that takes part in osteoblast commitment is Osterix (Osx) [ ], whose expression may or may not be dependent on Runx2 transcriptional activity. Osx promotes the expression of Satb2, a gene coding for a transcription factor belonging to the family of special AT-rich binding proteins [ ]. Characterization of the bone phenotype of Satb2 knockout (KO) mice revealed the involvement of Satb2 in osteoblast differentiation [ ], while Satb2 haploinsufficiency is causative of craniofacial defects in humans [ ]. The transcription factor complex crucial for osteogenesis also includes activating transcription factor (ATF), whose deletion in mice impairs collagen I synthesis, and thereby eventually leads to a low bone mass phenotype [ ].

Once osteoprogenitors become Alp-positive preosteoblasts, they undergo morphological changes, acquiring a cuboidal shape and starting to synthesize bone matrix proteins. A mature osteoblast is a terminally differentiated, polarized cell, characterized by a high expression of bone matrix proteins like osteocalcin (OCN), bone sialoprotein (BSP) I and II, osteopontin (OPN), and collagen type I, in addition to the ability to secrete nodules of mineralization.

Several systemic, paracrine, and autocrine factors contribute to orchestrate the process of osteoblast differentiation. Among these factors, we will describe the wingless-type MMTV integration site family (WNT) and bone morphogenic proteins (BMPs) signaling pathways in more detail since a crucial role emerged for these molecules in osteomimicry.

The WNT family includes at least 19 secreted glycoproteins encompassing a broad range of functions, especially during embryonic development [ ] and under pathological conditions, such as in cancer [ , ]. In bone, WNT promotes osteogenic functions by means of canonical or noncanonical pathways [ ]. In the former (i.e., WNT/β-catenin signaling pathway), WNT molecules interact with the receptor Frizzled (FZD) and low-density lipoprotein (LDL) receptor-related protein (LRP)5/6 co-receptors, thus preventing the phosphorylation of β-catenin induced by glycogen synthase kinase 3β (GSK3β). In the hypophosphorylated form, β-catenin is more stable and can translocate to the nucleus, where it promotes the transcription of osteoblast-differentiating genes. In contrast, when β-catenin is phosphorylated it undergoes proteasomal degradation, hence failing to initiate transcription of osteogenic genes. The noncanonical WNT/planar cell polarity (PCP) pathway also partakes in osteoblast differentiation, with Wnt-5a exerting its osteogenic effect by binding to FZD and to receptor tyrosine kinase-like orphan receptor (ROR) 1/2 [ ]. This results in the activation of c-jun NH2-terminal kinase (JNK) via disheveled (DVL) and the Rac and Rho small GTPases. A third WNT activation pathway is the noncanonical WNT/Ca ²⁺ pathway, in which WNT binding to FZD results in intracellular calcium (Ca ²⁺ ) release. This activates a number of Ca ²⁺ -dependent enzymes, such as protein kinase C (PKC), calcineurin, and calmodulin-dependent protein kinase II (CamKII) [ ].

Of great interest for the physiopathology of the bone was the identification of specific molecules inhibiting the WNT pathway, such as sclerostin (SOST), secreted FZD-related protein (sFRP), and Dickkopf (DKK) 1. SOST is a glycoprotein mainly produced by osteocytes, which binds to LRP5/6 and LRP4, thus acting as an extracellular antagonist [ , ]. Loss-of-function mutations of SOST are causative of the high bone mass diseases sclerosteosis type I and van Buchem disease [ , ]. The sFRP family includes five members acting as decoy receptors for WNT ligands [ ], and mutations in sFRP4 have been associated with skeletal disorders [ ]. DKK1 is a secreted glycoprotein expressed by mature osteoblasts and osteocytes, which binds to LRP5, thus inhibiting the WNT/β-catenin pathway. Indeed, mice lacking DKK1 showed increased bone mass [ ], while DKK1-overexpressing mice are osteopenic due to reduced osteoblast numbers [ ].

BMPs belong to the transforming growth factor β (TGF-β) family, which also includes TGF-β1, TGF-β2, and TGF-β3 and activins, all regulating osteoblastogenesis [ ]. Most BMPs are osteogenic, such as BMP-2, BMP-4, BMP-6, and BMP-7, as demonstrated by the increase in the expression and activity of Alp in preosteoblasts and of OCN in mature osteoblasts after treatment with these molecules [ ]. BMPs also stimulate the formation of nodules of mineralization [ ]. In contrast, BMP-3A and BMP-3b abrogate osteoblast differentiation [ , ]. BMPs regulate osteoblastogenesis by activating an intracellular pathway involving SMAD proteins, which in turn favor the activation of the transcription factors Runx2 and Osx. An alternative signaling pathway promoted by BMPs leads to the activation of mitogen activated protein kinase (MAPK) pathway, which promotes the expression of Alp and OCN [ ].

Other transcription factors involved in osteoblast differentiation that have emerged more recently are Forkhead box O (FOXO) and activating transcription factor 4 (ATF4) [ , ]. It has been shown that FOXO1 interacts with ATF4 to stimulate bone matrix mineralization and OCN expression [ ].