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Forkhead box O1 (FOXO1) is upregulated during bone formation and in response to stimulation by bone morphogenetic proteins. Studies presented here examined the functional role of FOXO1 in a well defined culture system in which pre-osteoblastic cells undergo terminal differentiation in vitro. Mineralizing cultures of MC3T3-E1 cells were examined with or without FOXO1 knockdown by RNAi. Normal cells show the upregulation of FOXO1 and RUNX2 DNA binding activity, alkaline phosphatase activity, and mRNA levels of FOXO1, RUNX2, type 1 collagen, osteocalcin and MMP13 during formation of mineralizing nodules. In FOXO1 depleted cells each of these measurements was significantly reduced compared to values in control cells transfected with scrambled siRNA (P

To gain insight into the effect of diabetes on fracture healing, experiments were carried out focusing on chondrocyte apoptosis during the transition from cartilage to bone. Type 1 diabetes was induced in mice by multiple low-dose streptozotocin injections, and simple transverse fractures of the tibia or femur was carried out. Large-scale transcriptional profiling and gene set enrichment analysis were performed to examine apoptotic pathways on total RNA isolated from fracture calluses on days 12, 16, and 22, a period of endochondral bone formation when cartilage is resorbed and chondrocyte numbers decrease. Tumor necrosis factor α (TNF-α) protein levels were assessed by ELISA and caspase-3 by bioactivity assay. The role of TNF was examined by treating mice with the TNF-specific inhibitor pegsunercept. In vitro studies investigated the proapoptotic transcription factor FOXO1 in regulating TNF-induced apoptosis of chondrogenic ATDC5 and C3H10T1/2 cells as representative of differentiated chondrocytes, which are important during endochondral ossification. mRNA profiling revealed an upregulation of gene sets related to apoptosis in the diabetic group on day 16 when cartilage resorption is active but not day 12 or day 22. This coincided with elevated TNF-α protein levels, chondrocyte apoptosis, enhanced caspase-3 activity, and increased FOXO1 nuclear translocation (p < .05). Inhibition of TNF significantly reduced these parameters in the diabetic mice but not in normoglycemic control mice (p < .05). Silencing FOXO1 using siRNA in vitro significantly reduced TNF-induced apoptosis and caspase activity in differentiated chondrocytes. The mRNA levels of the proapoptotic genes caspase-3, caspase-8, caspase-9, and TRAIL were significantly reduced with silencing of FOXO1 in chondrocytic cells. Inhibiting caspase-8 and caspase-9 significantly reduced TNF-induced apoptosis in chondrogenic cells. These results suggest that diabetes causes an upregulation of proapoptotic genes during the transition from cartilage to bone in fracture healing. Diabetes increased chondrocyte apoptosis through a mechanism that involved enhanced production of TNF-α, which stimulates chondrocyte apoptosis and upregulates mRNA levels of apoptotic genes through FOXO1 activation. © 2010 American Society for Bone and Mineral Research.

Osteoimmunolgy involves the interaction of the immune system with skeletal elements. This interaction can lead to the formation of osseous lesions. To investigate how the acquired immune response could contribute to osteolytic lesions, we injected the periodontal pathogen Porphyromonas gingivalis adjacent to calvarial bone with or without prior immunization against the bacterium. Activation of the acquired immune response increased osteoclastogenesis and decreased coupled bone formation. The latter was accompanied by an increase in nuclear translocation of the transcription factor FOXO1 in vivo, increased apoptosis of bone-lining cells measured by the TUNEL assay and number of activated caspase-3 positive cells and a decrease in bone lining cell density. Further studies were conducted with MC3T3 osteoblastic cells. Apoptosis and increased FOXO1 DNA binding activity were induced when a combination of cytokines was tested, IL-β, TNF-α, and IFN-γ. Knockdown of FOXO1 by small interfering RNA significantly reduced cytokine stimulated apoptosis, cleaved caspase-3/7 activity and decreased mRNA levels of the proapoptotic genes, TNF-α, FADD, and caspase-3, -8, and -9. These results indicate that activation of the acquired immunity by a periodontal pathogen reduces the coupling of bone formation and resorption. This may occur by enhancing bone lining cell apoptosis through a mechanism that involves increased FOXO1 activation. These studies give insight into inflammatory bone diseases such as periodontal disease and arthritis were the formation of lytic lesions occurs in conjunction with deficient bone formation and activation of an acquired immune response.

Bone mineral composition, crystallinity, and bone mineral content of osteoporotic patients are different from those of normal subjects. We review the evidence that these mineralization parameters contribute to the strength (fracture resistance) of bone and the methods that have been used to examine them. A specific example is provided from analysis of biopsies from the Multiple Outcomes in Raloxifene Evaluation trial. For the analyses, randomly selected biopsies from placebo, low-dose, and high-dose groups (n = 5 per group) obtained at time zero and 2 years after treatment were examined by infrared imaging spectroscopy. In all cases, comparable increases in mineral content were found, but there were no significant variations in mineral crystallinity.