Carnosic acid attenuates RANKL-induced oxidative stress and osteoclastogenesis via induction of Nrf2 and suppression of NF-κB and MAPK signalling

Costimulatory signals are required for activation of immune cells, but it is not known whether they contribute to other biological systems. The development and homeostasis of the skeletal system depend on the balance between bone formation and resorption. Receptor activator of NF-kappaB ligand (RANKL) regulates the differentiation of bone-resorbing cells, osteoclasts, in the presence of macrophage-colony stimulating factor (M-CSF). But it remains unclear how RANKL activates the calcium signals that lead to induction of nuclear factor of activated T cells c1, a key transcription factor for osteoclastogenesis. Here we show that mice lacking immunoreceptor tyrosine-based activation motif (ITAM)-harbouring adaptors, Fc receptor common gamma subunit (FcRgamma) and DNAX-activating protein (DAP)12, exhibit severe osteopetrosis owing to impaired osteoclast differentiation. In osteoclast precursor cells, FcRgamma and DAP12 associate with multiple immunoreceptors and activate calcium signalling through phospholipase Cgamma. Thus, ITAM-dependent costimulatory signals activated by multiple immunoreceptors are essential for the maintenance of bone homeostasis. These results reveal that RANKL and M-CSF are not sufficient to activate the signals required for osteoclastogenesis.

The receptor activator of NF-kappaB ligand (RANKL) induces osteoclast differentiation from bone marrow cells in the presence of macrophage colony-stimulating factor. We found that treatment of bone marrow cells with SB203580 inhibited osteoclast differentiation via inhibition of the RANKL-mediated signaling pathway. To elucidate the role of p38 mitogen-activated protein (MAP) kinase pathway in osteoclastogenesis, we employed RAW264 cells which could differentiate into osteoclast-like cells following treatment with RANKL. In a dose-dependent manner, SB203580 but not PD98059, inhibited RANKL-induced differentiation. Among three MAP kinase families tested, this inhibition profile coincided only with the activation of p38 MAP kinase. Expression in RAW264 cells of the dominant negative form of either p38alpha MAP kinase or MAP kinase kinase (MKK) 6 significantly inhibited RANKL-induced differentiation of the cells. These results indicate that activation of the p38 MAP kinase pathway plays an important role in RANKL-induced osteoclast differentiation of precursor bone marrow cells.

The proto-oncogene c-fos is the cellular homologue of v-fos originally isolated from murine osteosarcoma. Fos protein is a major component of the AP-1 transcription factor complex, which includes members of the jun family. Stable expression of c-fos in mice has been demonstrated in developing bones and teeth, haematopoietic cells, germ cells and in the central nervous system. It has been proposed that c-fos has an important role in signal transduction, cell proliferation and differentiation. We have previously demonstrated that overexpression of c-fos in transgenic and chimaeric mice specifically affects bone, cartilage and haematopoietic cell development. To understand better the function of c-fos in vivo, we used gene targeting in embryonic stem cells to generate cells and mice lacking c-fos. Here we report that heterozygous fos +/- mice appear normal, although females exhibit a distorted transmission frequency. All homozygous fos -/- mice are growth-retarded, develop osteopetrosis with deficiencies in bone remodelling and tooth eruption, and have altered haematopoiesis. These data define the c-Fos protein as an essential molecule for the development of specific cellular compartments.