Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis: a longitudinal study of gene expression in bone tissue from glucocorticoid-treated mice.

Glucocorticoids (GCs) are frequently prescribed for the treatment of many chronic noninfectious inflammatory disorders, including arthritis, pulmonary diseases, and skin diseases. Although GCs are potent antiinflammatory agents, long-term use results in several adverse side effects, the most common of which is bone loss, which increases the risk of fracture throughout the skeleton (1). Patients treated with GCs have been reported to have an early, rapid increase in bone resorption accompanied by a prolonged reduction in bone formation (2). The influence of GCs on bone resorption was thought to be indirect and related in part to reduced calcium absorption and increased renal calcium excretion (3). However, recent studies have shown that GCs act directly on osteoclasts to decrease apoptosis of mature osteoclasts (4). Kim et al (5) observed that GCs in vitro inhibited the proliferation of osteoclasts from bone marrow macrophages in a dose-dependent manner. In addition, higher GC doses had no effect on osteoclast maturation but inhibited the ability of osteoclasts to reorganize their cytoskeleton. Therefore, GC excess results in an increased number of osteoclasts but an apparent inhibition of function, with impaired spreading and degrading of mineralized matrix (5). GCs also alter osteoblast and osteocyte function that contributes to GC-induced osteoporosis (3). GCs directly inhibit cellular proliferation and differentiation of the osteoblast lineage (1), reduce osteoblast maturation and activity (6), and induce osteoblast and osteocyte apoptosis in vivo (7). The suppression of osteoblast function by GCs is reported to be associated with alteration of the Wnt signaling pathway (8), a critical pathway for osteoblastogenesis (9). GCs enhance expression of Dkk-1 (10), one of the Wnt antagonists that prevent soluble Wnt protein from binding to its receptor complex (11). GCs maintain levels of glycogen synthase kinase 3 (GSK3β) (12), a key kinase phosphorylating β-catenin, thereby preventing the translocation of β-catenin into the nucleus and the initiation of transcription in favor of osteoblastogenesis. GCs may also enhance bone marrow stromal cell development toward adipocyte lineage rather than toward osteoblasts (13). Moreover, the loss of osteocytes by GC-induced apoptosis (14) may disrupt the osteocyte–canalicular network, resulting in a failure to direct bone remodeling at the trabecular surface. The GC-induced changes in osteocyte function also result in weakening of the localized material properties around osteocytes as well as whole-bone strength (15). Information regarding the majority of molecular mechanisms responsible for GC excess was derived from in vitro studies of individual cell lines. Therefore, we hypothesized that expression of osteoblast and osteoclast genes obtained from the long bones of male mice treated with GCs would change over time. To test this hypothesis, we used microarray technology and verified our microarray data by real-time polymerase chain reaction (PCR) analysis of selected genes. In addition, we evaluated the association of gene transcription with changes in bone turnover and whole-bone structural changes in GC-treated mice, and we identified sequential alteration of gene expression in osteoclastogenesis, adipogenesis, and osteogenesis. These data provide in vivo evidence supporting direct or indirect regulation of many new gene transcripts associated with GC excess and enhance our understanding of GC-induced bone loss.