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

Abstract

Objective: Glucocorticoid (GC) excess induces alterations in bone metabolism that weaken bone structure and increase fracture risk. The aim of this study was to identify genes associated with bone metabolism in GC-treated mice, by performing a microarray analysis.

Methods: Long bones from mice exposed to GC excess were collected after 0, 7, 28, and 56 days of treatment, to measure bone microarchitecture and extract RNA for microarray analyses.

Results: Bone loss in this animal model was confirmed by changes in bone turnover markers as well as bone architecture, as measured by microfocal computed tomography. GC excess induced an early up-regulation of genes involved in osteoclast activation, function, and adipogenesis, which peaked on day 7. The expression of genes associated with osteoclast cytoskeletal reorganization and genes associated with matrix degradation peaked on day 28. On day 28 and day 56, the expression of genes associated with osteoblast activation and maturation was decreased from baseline, while the expression of Wnt antagonists was increased. In addition, the expression of genes expressed in osteocytes associated with bone mineralization was significantly higher at the later time points, day 28 and day 56. Reverse transcription-polymerase chain reaction confirmed the results of microarray analysis in selected genes.

Conclusion: GC excess is associated with early activation of genes associated with osteoclastogenesis and adipogenesis and a later suppression of genes associated with osteogenesis and mineralization. Novel interventions with agents that modulate either Wnt signaling or mineralization may be effective in GC-induced osteoporosis.

Figure 1

Time-dependent changes in bone turnover markers and bone microarchitecture associated with glucocorticoid (GC) excess. a, Levels of C-telopeptide of type I collagen (CTX), a marker for osteoclast resorption, showing an increase of ~40% on days 7 and 28 in GC-treated mice compared with placebo (PL)–treated mice. b, Levels of osteocalcin, showing a decrease of ~50% on days 28 and 56 in GC-treated mice compared with placebo-treated mice. c–e, Trabecular bone volume, as measured by repeated in vivo microfocal computed tomography at the distal femurs. The volume decreased by nearly 20% from day 0 to day 28 and then leveled off. Values in a–c are the mean and SD. a = P < 0.05 versus day 0; b = P < 0.05 versus placebo at the same time point.

Figure 2

Time-dependent gene profiling of integrin-mediated signaling associated with glucocorticoid excess. a, Heat map of the integrin-mediated pathway. Red indicates up-regulation, and green indicates down-regulation compared with day 0. Up-regulated genes included αv integrin, αM integrin, β3 integrin, and Vav3; up-regulation was at its highest level on day 28. Down-regulated genes included β1 integrin, β4 integrin, α5 integrin, β6 integrin, β7 integrin, ADAM-17, and ADAM-33. b, Main biologic Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of integrin-mediated signaling. ECM = extracellular matrix.

Figure 3

Time-dependent gene profiling of matrix proteolysis associated with glucocorticoid excess. a, Heat map of the matrix proteolysis pathway. Red indicates up-regulation, and green indicates down-regulation compared with day 0. Up-regulated genes included serine proteases 22 and 35, matrix metalloproteinase 8 (MMP-8), MMP-9, MMP-12, MMP-19, and cathepsins S, H, K, G, and C. b, Main biologic Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of matrix proteolysis.

Figure 4

Time-dependent gene profiling of Wnt receptor signaling associated with glucocorticoid excess. a, Heat map of the Wnt receptor signaling pathway. Red indicates up-regulation, and green indicates down-regulation compared with day 0. Up-regulated genes included Wnt inhibitors such as Dkk-1 and Wnt inhibitory factor 1. Down-regulated genes included Lrp1, Dsh1, Axin1, and Frizzled 7; down-regulation was most significant on either day 28 or day 56. b, Main biologic Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of Wnt receptor signaling.

Figure 5

Time-dependent gene profiling of bone remodeling associated with glucocorticoid excess. a, Heat map of the bone remodeling pathway. Red indicates up-regulation, and green indicates down-regulation compared with day 0. Up-regulated genes included Dmp1, Phex, Csf1, and Sost; up-regulation was most significant on days 28 and 56. Down-regulated genes included Bmp2 and TGFB1; down-regulation was most significant on day 28 for TGFB1 and day 56 for Bmp2. b, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of bone remodeling overlapped with the above biologic pathways, as in Figures 2–4. TGF = transforming growth factor.

Figure 6

Time course of changes in the expression of selected genes involved in osteoclastogenesis, in association with glucocorticoid excess, as determined by polymerase chain reaction. The expression of Csf1, Adam8, and Mmp9, which was significantly up-regulated on day 7, remained ~2-fold higher than the day 0 level until day 56. The expression of Wnt inhibitors, including Dkk-1, Sost, and WNF1, was increased from day 28 to day 56. Expression of the gene that represented osteoblast differentiation, Akp2, was suppressed throughout the study. The level of Dmp1, which is expressed by osteoblasts and osteocytes, was increased on days 28 and 56. a = P < 0.05 versus day 0.