TGF-ß in jaw tumor fluids induces RANKL expression in stromal fibroblasts.

Odontogenic tumors and cysts, arising in the jawbones, grow by resorption and destruction of the jawbones. However, mechanisms underlying bone resorption by odontogenic tumors/cysts remain unclear. Odontogenic tumors/cysts comprise odontogenic epithelial cells and stromal fibroblasts, which originate from the developing tooth germ. It has been demonstrated that odontogenic epithelial cells of the developing tooth germ induce osteoclastogenesis to prevent the tooth germ from invading the developing bone to maintain its structure in developing bones. Thus, we hypothesized that odontogenic epithelial cells of odontogenic tumors/cysts induce osteoclast formation, which plays potential roles in tumor/cyst outgrowth into the jawbone. The purpose of this study was to examine osteoclastogenesis by cytokines, focusing on transforming growth factor-ß (TGF-ß), produced by odontogenic epithelial cells. We observed two pathways for receptor activator of NF-κB ligand (RANKL) induction by keratocystic odontogenic tumor fluid: the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway through interleukin-1α (IL-1α) signaling and non-COX-2/PGE2 pathway through TGF-ß receptor signaling. TGF-ß1 and IL-1α produced by odontogenic tumors/cysts induced osteoclastogenesis directly in the osteoclast precursor cells and indirectly via increased RANKL induction in the stroma.

Signaling of extracellular inorganic phosphate up-regulates cyclin D1 expression in proliferating chondrocytes via the Na+/Pi cotransporter Pit-1 and Raf/MEK/ERK pathway.

As chondrocytes mature, the concentration of inorganic phosphate (Pi) increases in the extracellular milieu. It was demonstrated that the progressive accumulation of Pi started from the proliferative zone and peaked in the hypertrophic zone of growth plate. Although extracellular Pi is reported to be involved in the apoptosis and mineralization of mature chondrocytes, its role in proliferating chondrocytes remains unclear. Here we investigated this role utilizing ATDC5, an established cell model of chondrocytic differentiation. In proliferating ATDC5 cells, we found that the expression of cyclin D1 was up-regulated, and that of alkaline phosphatase (ALP) was down-regulated in response to an increase in extracellular Pi within 24h. Moreover, an increase in extracellular Pi-induced activation of the Raf/MEK/ERK pathway, and treatment with a MEK inhibitor PD98059 abolished the effects on the expression of cyclin D1 and ALP, indicating that extracellular Pi regulates the expression of these genes through the Raf/MEK/ERK pathway. Consistent with its up-regulation of cyclin D1 expression, the extracellular Pi facilitated the proliferation of ATDC5 cells. Treatment with phosphonoformic acid (PFA), an inhibitor of sodium/phosphate (Na(+)/Pi) cotransporters, abrogated the activation of the Raf/MEK/ERK pathway and gene expression induced by the increase in extracellular Pi. Knocking down of the type III Na(+)/Pi cotransporter Pit-1 diminished the responsiveness of ATDC5 cells to the increase in extracellular Pi. Interestingly, the increased extracellular Pi induced the phosphorylation of fibroblast growth factor receptor substrate 2α (FRS2α), which was also cancelled by knocking down of the expression of Pit-1. In primary chondrocytes isolated from mouse rib cages as well, increased extracellular Pi induced the phosphorylation of ERK1/2 and alterations in the expression of cyclin D1 and ALP, both of which were abolished by treatment with PFA. These results suggest that signaling by extracellular Pi is mediated by Pit-1 and FRS2α, and leads to activation of the Raf/MEK/ERK pathway and increased expression of cyclin D1, which facilitates the proliferation of immature chondrocytes.

Involvement of nuclear factor I transcription/replication factor in the early stage of chondrocytic differentiation.

Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. To identify the genes involved in chondrocytic differentiation, we applied this method to a murine mesenchymal cell line, ATDC5, which differentiate into mature chondrocytes in the presence of insulin, and isolated a clone in which the gene encoding a transcription/replication factor, nuclear factor I-B (NFIB), was trapped. In this particular clone, named #7-57, the trap vector pPT1-geo was inserted into intron 6 of the NFIB gene in one of the alleles. As a result, both wild-type NFIB and a mutant protein lacking the carboxyl-terminal transactivation/repression domain were expressed in the clone. Immunoprecipitation/Western blotting confirmed the interaction between wild-type NFIB and the truncated protein derived from the trapped allele, suggesting that the mutant protein formed a heterodimer with wild-type NFI proteins. When cultured in the differentiation medium, #7-57 exhibited impaired nodule formation and less accumulation of cartilageous matrices compared with the parental ATDC5 cells. In addition, the expression of marker genes for proliferating chondrocytes, including type II collagen (Col2a1), matrillin-1, and PTHrP, was reduced in the clone. The expression of SOX9 was also slightly decreased in the clone #7-57 compared with the parental cells. The overexpression of wild-type NFIB in parental ATDC5 cells resulted in the increased expression of Col2a1, and a series of reporter assays using a Col2a1 promoter/enhancer-luciferase construct demonstrated the transcriptional regulation of the gene by NFIB and the dominant-negative effect of the truncated mutant derived from the trapped allele. Interestingly, mutation in the SOX9-binding site in the 48-bp cis-element located in intron 1 failed to abolish the transactivation of Col2a1 gene by NFIB, suggesting that NFI regulates the transactivation of Col2a1, at least in part, independently of SOX9. These results indicate the critical roles of NFI family transcription/replication factors in the early stage of chondrocytic differentiation.