Glucocorticoids (GCs), which play an important role in the normal regulation of bone remodeling, are widely used as anti-inflammatory and chemotherapeutic agents. However, continued exposure to GCs results in osteoporosis, which is partially due to apoptosis of osteoblasts and osteocytes. To understand the mechanism of how GCs induce cell death in osteoblasts, we examined apoptotic effects of dexamethasone (Dex), GC, on MC3T3-E1 osteoblast cells. Results revealed that Dex-induced apoptosis was inhibited by a GC receptor antagonist, mifepristone, and a general caspase inhibitor, Z-VAD-fmk, indicating that Dex induces apoptosis of MC3T3-E1 cells through the pathways involved in GC receptor and caspase. Glycogen synthase kinase 3b (GSK3b) is known to participate in apoptosis signaling in MC3T3-E1 cells. Dex activated both GSK3b and p38-mitogen-activated protein kinase (MAPK). The inhibition of GSK3b by inhibitor (LiCl) or small interference RNA (siRNA) decreased apoptosis. In contrast, the inhibition of p38-MAPK by inhibitor (SB203580) or siRNA did not decrease, but increase apoptosis. These results suggest that Dex-mediated apoptosis of osteoblasts is facilitated by GSK3b, but prevented by p38-MAPK.

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This study was performed to discover a novel herbal therapeutic for effective glucocorticoid-induced osteoporosis (GIO) treatment and further to clarify its molecular mechanism of action. Ethanol or methanol extracts of 68 edible Korean native plants were screened to find effective natural plant sources for the treatment of GIO, and Poncirus trifoliata (L.) (Rutaceae, PT) was selected as a final candidate because of its high inhibitory activity plus its novelty. The hexane extract of PT (PT-H) inhibited apoptotic cell death in dexamethasone-induced osteoblastic cell lines, C3H10T1/2 and MC3T3-E1. In vivo mouse results indicated that PT-H not only had an inhibitory effect on the bone loss caused by glucocorticoid, but also promoted bone formation. The molecular mechanisms behind the effect of PT-H on GIO were further clarified by screening of differentially expressed genes (DEGs) between dexamethasone (Dex)-induced osteoblastic cells with or without PT-H treatment. Finally, it was found that the expression level of AnxA6 in Dex-induced osteoblastic cells and prednisolone (PD)-treated GIO-model mice was significantly decreased by PT-H treatment. These findings suggest that PT-H has a strong in vitro and in vivo inhibitory effect on GIO, and decreased expression of AnxA6 may play a key role in this inhibition.

Poncirin, a flavonoid isolated from the fruit of Poncirus trifoliata, possesses anti-bacterial and anti-inflammatory activities. However, the action of poncirin in bone biology is unclear. In this study, the in vivo and in vitro effects of poncirin in a glucocorticoid-induced osteoporosis (GIO) mouse model were investigated. Seven-month-old male mice were assigned to the following five groups: (1) sham-implantation (sham), (2) prednisolone 2.1 mg/kg/day (GC), (3) GC treated with 10 mg/kg/day of genistein, (4) GC treated with 3 mg/kg/day of poncirin, (5) and GC treated with 10 mg/kg/day of strontium (GC + SrCl(2)). After 8 weeks, bone loss was measured by microcomputed tomography. Osteocalcin (OC) and C-terminal telopeptides of type I collagen (CTX) were evaluated in sera. Runx2 protein, OC and osteoprotegerin (OPG) mRNA expression, alkaline phosphatase (ALP) activity, and mineral nodule assay were performed in C3H10T1/2 or primary bone marrow stromal cells. Poncirin significantly increased the bone mineral density and improved the microarchitecture. Poncirin increased serum OC, Runx2 protein production, expression of OC and OPG mRNA, ALP activity, and mineral nodule formation; and decreased serum CTX. These effects were more prominent in the poncirin group compared to the other positive control groups (genistein and strontium). The poncirin-mediated restoration of biochemical bone markers, increased bone mineral density, and improved trabecular microarchitecture likely reflect increased bone formation and decreased bone resorption in GIO mice.

Glycogen synthase kinase-3beta (GSK3beta) controls the survival of osteoblasts during bone development through Wnt canonical signaling. GSK3beta is a key factor for osteoblastogenesis, but relatively less is known regarding its role in osteoblast apoptosis. Genotoxic stress induced by etoposide promoted apoptotic signaling by GSK3beta activation in C3H10T1/2 cells, a mouse mesenchymal cell line. Etoposide led to the time-dependent activation of GSK3beta and caspase-3, which resulted in PARP cleavage. LiCl (a specific inhibitor) and siRNA (gene knock-down) of GSK3beta prevented the effects of etoposide on apoptosis. Staurosporine also induced apoptosis in C3H10T1/2 cells, but LiCl could not rescue. Bcl-2 was decreased in the cells by exposure to etoposide. LiCl completely recovered Bcl-2 expression as shown by both the mRNA and the protein expression levels. In conclusion, etoposide-induced apoptosis in C3H10T1/2 cells is mediated by GSK3beta, which leads to caspase-3 activation via decrease in Bcl-2 expression.

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