Activation function 2 (AF2) domain of estrogen receptor-α regulates mechanotransduction during bone fracture healing in estrogen-competent mice.

External mechanostimulation applied by whole-body low-magnitude high-frequency vibration (LMHFV) was demonstrated to cause no or negative effects on fracture healing in estrogen-competent rodents, while in ovariectomized (OVX), estrogen-deficient rodents bone formation after fracture was improved. Using mice with an osteoblast-specific deletion of the estrogen receptor α (ERα), we demonstrated that ERα signaling in osteoblasts is required for both the anabolic and catabolic effects of LMHFV during bone fracture healing in OVX and non-OVX mice, respectively. Because the vibration effects mediated by ERα were strictly dependent on the estrogen status, we hypothesized different roles of ligand-dependent and -independent ERα signaling. To investigate this assumption in the present study, we used mice with a deletion of the C-terminal activation function (AF) domain-2 of the ERα receptor, which mediated ligand-dependent ERα signaling (ERαAF-20). OVX and non-OVX ERαAF-20 animals were subjected to femur osteotomy followed by vibration treatment. We revealed that estrogen-competent mice lacking the AF-2 domain were protected from LMHFV-induced impaired bone regeneration, while the anabolic effects of vibration in OVX mice were not affected by the AF-2 knockout. RNA sequencing further showed that genes involved in Hippo/Yap1-Taz and Wnt signaling were significantly downregulated upon LMHFV in the presence of estrogen in vitro. In conclusion, we demonstrated that the AF-2 domain is crucial for the negative effects of vibration during bone fracture healing in estrogen-competent mice suggesting that the osteoanabolic effects of vibration are rather mediated by ligand-independent ERα signaling.

Osteoblast lineage Sod2 deficiency leads to an osteoporosis-like phenotype in mice.

Osteoporosis is a systemic metabolic skeletal disease characterized by low bone mass and strength associated with fragility fractures. Oxidative stress, which results from elevated intracellular reactive oxygen species (ROS) and arises in the aging organism, is considered one of the critical factors contributing to osteoporosis. Mitochondrial (mt)ROS, as the superoxide anion (O2-) generated during mitochondrial respiration, are eliminated in the young organism by antioxidant defense mechanisms, including superoxide dismutase 2 (SOD2), the expression and activity of which are decreased in aging mesenchymal progenitor cells, accompanied by increased mtROS production. Using a mouse model of osteoblast lineage cells with Sod2 deficiency, we observed significant bone loss in trabecular and cortical bones accompanied by decreased osteoblast activity, increased adipocyte accumulation in the bone marrow and augmented osteoclast activity, suggestive of altered mesenchymal progenitor cell differentiation and osteoclastogenesis. Furthermore, osteoblast senescence was increased. To date, there are only a few studies suggesting a causal association between mtROS and cellular senescence in tissue in vivo. Targeting SOD2 to improve redox homeostasis could represent a potential therapeutic strategy for maintaining bone health during aging.

In Vitro Characterization of Doxorubicin-Mediated Stress-Induced Premature Senescence in Human Chondrocytes.

Accumulation of senescent chondrocytes is thought to drive inflammatory processes and subsequent cartilage degeneration in age-related as well as posttraumatic osteoarthritis (OA). However, the underlying mechanisms of senescence and consequences on cartilage homeostasis are not completely understood so far. Therefore, suitable in vitro models are needed to study chondrocyte senescence. In this study, we established and evaluated a doxorubicin (Doxo)-based model of stress-induced premature senescence (SIPS) in human articular chondrocytes (hAC). Cellular senescence was determined by the investigation of various senescence associated (SA) hallmarks including ß-galactosidase activity, expression of p16, p21, and SA secretory phenotype (SASP) markers (IL-6, IL-8, MMP-13), the presence of urokinase-type plasminogen activator receptor (uPAR), and cell cycle arrest. After seven days, Doxo-treated hAC displayed a SIPS-like phenotype, characterized by excessive secretion of SASP factors, enhanced uPAR-positivity, decreased proliferation rate, and increased ß-galactosidase activity. This phenotype was proven to be stable seven days after the removal of Doxo. Moreover, Doxo-treated hAC exhibited increased granularity and flattened or fibroblast-like morphology. Further analysis implies that Doxo-mediated SIPS was driven by oxidative stress as demonstrated by increased ROS levels and NO release. Overall, we provide novel insights into chondrocyte senescence and present a suitable in vitro model for further studies.