Osteoclast-derived complement component 3a stimulates osteoblast differentiation.

Bone remodeling is regulated by a coupling of resorption to subsequent formation; however, the "coupling factor" and underlying mechanism are not fully understood. Here, we found that the condition medium (CM) of mature osteoclasts contains a humoral factor that stimulates the differentiation of primary osteoblasts, as determined by alkaline phosphatase (ALP) activity. We purified osteoblastogenesis-stimulating activity from 3 L of osteoclast CM through successive ion exchange chromatographies by monitoring the ALP activity of osteoblasts and identified complement component 3 (C3). Expression of the C3 gene increased during osteoclastogenesis, and the cleavage product C3a was detected by ELISA in the CM of osteoclasts but not in that of bone marrow macrophages. The osteoblastogenesis-stimulating activity present in osteoclast CM was inhibited by a specific antagonist of the C3a receptor (C3aR), SB290157. Conversely, the retroviral expression of C3a as well as treatment with the C3aR agonist, benzeneacetamide, stimulated osteoblast differentiation. C3 gene expression in bone was increased in the high bone turnover states of ovariectomy (OVX) or a receptor activator of NF-κB ligand (RANKL) injection, and blocking the action of C3a with the daily administration of SB290157 resulted in the attenuation of bone formation elevated by OVX and the exacerbation of bone loss. These results suggest that osteoclast-derived C3a functions in the relay from bone resorption to formation and may be a candidate for a coupling factor.

Osteoclast-secreted CTHRC1 in the coupling of bone resorption to formation.

Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-ß, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule serves as a coupling factor for bone resorption to formation. We found that CTHRC1, a protein secreted by mature bone-resorbing osteoclasts, targets stromal cells to stimulate osteogenesis. Cthrc1 expression was robustly induced when mature osteoclasts were placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increased in a high-turnover state (such as that induced by RANKL injections in vivo), but decreased in conditions associated with suppressed bone turnover (such as with aging and after alendronate treatment). Targeted deletion of Cthrc1 in mice eliminated Cthrc1 expression in bone, whereas its deficiency in osteoblasts did not exert any significant effect. Osteoclast-specific deletion of Cthrc1 resulted in osteopenia due to reduced bone formation and impaired the coupling process after resorption induced by RANKL injections, impairing bone mass recovery. These data demonstrate that CTHRC1 is an osteoclast-secreted coupling factor that regulates bone remodeling.

Transcriptional repression of the mouse wee1 gene by TBP-related factor 2.

TBP-related factor 2 (TRF2), one of the TBP family proteins, is involved in various cellular functions through its transcription stimulation activity. We previously reported that TRF2 is involved in reduction of wee1 mRNA in genotoxin-treated chicken cells. In this study, we investigated the role of TRF2 in wee1 gene expression. It was found that wee1 mRNA was decreased in hydroxyurea-treated NIH3T3 cells. Mouse wee1 promoter activity was repressed by TRF2 in mouse and chicken cells. Chromatin immunoprecipitation and plasmid immunoprecipitation analyses revealed that TRF2 is recruited to the wee1 promoter in accordance with the transcriptional repression. A mutant TRF2 that lacks TFIIA-binding capacity lost its repressive function. This mutant was less recruited to the wee1 promoter than was the wild-type one, and provided a decline in promoter-recruited TFIIA. Data in this study suggest that transcription repressive activity of TRF2 to wee1 promoter needs association with the promoter and TFIIA.

TATA-binding protein-related factor 2 is localized in the cytoplasm of mammalian cells and much of it migrates to the nucleus in response to genotoxic agents.

TBP (TATA-binding protein)-related factor 2 (TRF2) regulates transcription during a nuber of cellular processes. We previously demonstrated that it is localized in the cytoplasm and is translocated to the nucleus by DNA-damaging agents. However, the cytoplasmic localization of TRF2 is controversial. In this study, we reconfirmed its cytoplasmic localization in various ways and examined its nuclear migration. Stresses such as heat shock, redox agents, heavy metals, and osmotic shock did not affect localization whereas genotoxins such as methyl methanesulfonate (MMS), cisplatin, etoposide, and hydroxyurea caused it to migrate to the nucleus. Adriamycin, mitomycin C and gamma-rays had no obvious effect. We determined optimal conditions for the nuclear migration. The proportions of cells with nuclei enriched for TRF2 were 25-60% and 5-10% for stressed cells and control cells, respectively. Nuclear translocation was observed after 1 h, 4 h and 12 h for cisplatin, etoposide and MMS and hydroxyurea, respectively. The association of TRF2 with the chromatin and promoter region of the proliferating cell nuclear antigen (PCNA) gene, a putative target of TRF2, was increased by MMS treatment. Thus TRF2 may be involved in genotoxin-induced transcriptional regulation.