Glucocorticoids decrease the number and the function of osteo blasts. These effects lead to a suppression of bone formation, a central feature of the pathogenesis of GIO. Glucocorticoids decrease the replication of cells of the osteoblastic lineage, reducing the pool of cells that may differentiate into mature osteoblasts. The mechanism by which this occurs may relate to redirecting multipotential bone marrow stromal cells into the adipocyte line and away from osteoblast lineage precursors. Thus, glucocorticoids impair osteoblastic differentiation and maturation at the early stages of osteo blast development.
Mechanisms involved in this redirection of stromal cells include induction of nuclear factors of the CAAT enhancer binding protein family and the induction of peroxisome proliferator- activated receptor γ 2 (PPARγ 2), both of which play essential roles in adipogenesis.
A fundamental mechanism by which glucocorticoids inhibit osteoblast cell differentiation is by opposing Wnt/ β- catenin signalling. Wnt signalling has emerged as a key regulator of osteoblastogenesis and osteoclastogenesis. In skeletal cells, the canonical Wnt/ β- catenin signalling pathway operates. When Wnt is absent (or inhibited), β- catenin is phosphorylated by glycogen- synthase kinase- 3β (GSK- 3β), and then degraded by ubiquitination in the cytoplasm, thus preventing translocation into the nucleus. The binding of Wnt to specific receptors, called Frizzled, and to coreceptors, such as low- density lipoprotein receptor related proteins (LRP)- 5 and - 6, leads to an inhibition of GSK- 3β activity. When GSK- 3β is not active, stabilized β- catenin is able to translocate to the nucleus, where it associates with transcription factors to regulate gene expression. Inactivation of either Wnt or Ctnnb (encoding for β- catenin) results in impairment of osteoblastogenesis, and increased osteoclastogenesis, since canonical Wnt signalling results in a direct inhibitory effect on osteoclast progenitors and induction of osteoprotegerin (OPG) production. The Wnt pathway can be opposed by Wnt antagonists, such as Dickkopf-1 and sclerostin that prevent Wnt from binding to its receptor complex. Glucocorticoids enhance Dickkopf-1 and sclerostin expression, and maintain GSK 3- β in an active state, leading ultimately to the inactivation of β- catenin. The importance of sclerostin in mediating the effects of glucocorticoids on bone formation is emphasized by the demonstration, in mice with sclerostin deficiency, that bone integrity is maintained in the presence of glucocorticoid excess.
Glucocorticoids also suppress bone morphogenetic protein (BMP) signalling in osteoblasts either directly or indirectly by microRNAs which are small endogenous single- stranded non- coding ribonucleic acids (RNAs) that repress gene expression and bind to mRNA. BMPs are members of the transforming growth factor β (TGF- β) superfamily of polypeptides, which includes TGF- βs, activins, and inhibins. BMPs signal through the small molecules against decapentaplegic (SMADs) and the mitogen activated protein kinase signalling pathways.
Notch is a family of transmembrane receptors regulating cell fate decisions. In immature cells of the osteoblastic lineage, Notch suppresses cell differentiation by inhibiting Wnt signal ling and by interacting with Runx2 to prevent osteoblast maturation. Although glucocorticoids stimulate Notch1 and Notch2 expression in osteoblasts, Notch signalling is not induced by glucocorticoids.
In addition to inhibiting the differentiation of osteoblasts, glucocorticoids inhibit the function of the differentiated mature cells directly, and indirectly by suppressing the expression of insulin- like growth factor 1 (IGF1) by skeletal cells [31]. Glucocorticoids inhibit osteoblast- driven synthesis of type I collagen, the major component of the bone extracellular matrix, with a consequent decrease in bone matrix available for mineralization. The decrease in type I collagen synthesis occurs by transcriptional and post- transcriptional mechanisms.
Glucocorticoids have pro- apoptotic effects on osteoblasts and osteocytes due to activation of caspase 3, a common downstream effector of several apoptotic signalling pathways. Caspases are synthesized as proenzymes and are activated through auto catalysis or a caspase cascade. Active caspases contribute to apoptosis by cleaving target cellular proteins. Caspase 3 is a key mediator of apoptosis and is a common downstream effector of multiple apoptotic signalling pathways. The inhibitory effects of glucocorticoids on osteoblastic cell replication and differentiation and the increased apoptosis of mature osteoblasts, all contribute to the depletion of the osteoblastic cellular pool and decreased bone formation.
Osteocytes serve as mechanosensors, and play a role in the re pair of bone microdamage. Loss of osteocytes disrupts the osteocyte- canalicular network resulting in failure to detect signals that normally stimulate processes associated with the replacement of damaged bone. Disruption of the osteocyte- canalicular network can disrupt fluid flow within the network adversely affecting the material properties of the surrounding bone, independently of changes in bone remodelling or architecture. Glucocorticoids affect the function of osteocytes, by modifying the elastic modulus sur rounding osteocytic lacunae. Glucocorticoids induce the apoptosis of osteocytes. As a result, the normal maintenance of bone through this mechanism is impaired and the biomechanical properties of bone are compromised.
The initial bone loss occurring in patients exposed to glucocorticoids may be secondary to increased bone resorption. In fact, glucocorticoids increase the expression of receptor activator of NF- k B ligand (RANKL) and decrease the expression of its soluble decoy receptor, OPG in stromal and osteoblastic cells [20]. The combination of an increase in RANKL, a necessary signal for osteoclastogenesis, and a reduction in OPG, an inhibitor of RANKL action, leads to the initial phase of rapid bone loss. Glucocorticoids also enhance the ex pression of macrophage colony- stimulating factor (M- CSF), which in the presence of RANKL induces osteoclastogenesis. Moreover, glucocorticoids upregulate receptor subunits for osteoclastogenic cytokines of the gp130 family. Furthermore, glucocorticoids may decrease apoptosis of mature osteoclasts. Consequently, there is increased formation of osteoclasts with a prolonged life span ex plaining, at the cellular level, the enhanced bone resorption observed in the initial phases of GIO
