Mandibular condylar cartilage is the principal secondary cartilage, differing from primary cartilage in its rapid differentiation from progenitor cells (preosteoblasts/skeletoblasts) to hypertrophic chondrocytes. The expression of three transcription factors related to bone and cartilage formation, namely Runx2, Osterix and Sox9, was investigated at the onset of mouse mandibular condylar cartilage formation by in situ hybridization. Messenger RNAs for these three molecules were expressed in the condylar anlage, consisting of preosteoblasts/skeletoblasts, at embryonic day (E)14. Hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. Runx2 mRNA was expressed in the embryonic zone at the posterior position of the newly formed cartilage, in the bone collar and in the newly formed cartilage, but expression intensity in the newly formed cartilage was slightly weaker.Osterix mRNA was also expressed in the embryonic zone and in the bone collar, but was at markedly lower levels in the newly formed cartilage. Sox9 mRNA was continuously expressed from the embryonic zone to the newly formed cartilage. At this stage, Sox5 mRNA was expressed only in the newly formed cartilage. These results suggest that reduced expression of Osterix in combination with Sox9-Sox5 expression is important for the onset of condylar (secondary) cartilage formation.

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The expression of parathyroid hormone-related protein (PTHrP) was studied in a range of cell cultures representative of the osteoblast lineage and in rat calvarial sections. Primary newborn rat calvarial cells, a rat preosteoblastic cell line (UMR 201), a mouse stromal cell line (ST 2), a mouse calvaria-derived osteoblastic cell line (KS 4), and rat osteosarcoma cell lines (UMR 106-01 and -06), all expressed PTHrP when examined by reverse transcription polymerase chain reaction (RT-PCR). Using a radioimmunoassay we also demonstrated PTHrP in the conditioned medium of the cultured cells, with the exception of UMR 106-01 and -06 cells. Treatment of UMR 201 cells with all-trans-retinoic acid which induces them to acquire a more differentiated phenotype, also led to a time-dependent decrease in PTHrP mRNA levels as determined by RT-PCR, Northern blot analysis, and in situ hybridization. Decreased PTHrP levels in the conditioned medium of the treated cells was also observed. These results suggested that PTHrP production might be greater in less mature osteoblasts. Examination of the populations obtained from newborn rat calvariae by sequential collagenase digestion revealed that the early digests exhibited low ALP activity, low expression of PTH/PTHrP receptor mRNA, and no adenylate cyclase response to PTHrP(1-34). These populations showed the highest level of mRNA and production of PTHrP. Cells from later digests, the "osteoblast-rich" populations, had reduced PTHrP expression. Immunohistochemistry and in situ hybridization in sections of newborn rat calvariae showed PTHrP expression in cuboidal osteoblasts located adjacent to bone and in spindle-shaped cells in the periosteal region. It is concluded that PTHrP is produced by cells of the osteoblast lineage, supporting the hypothesis that PTHrP may function physiologically as a paracrine factor in bone.

Runx2 (runt-related transcription factor 2) deficient mice lacked the mandibular condylar cartilage and the mandibular bone. The anlage of the condylar process consisted of mesenchymal condensation, which expressed Type I collagen mRNA and alkaline phosphatase activity, but not Type II collagen and aggrecan mRNAs. Therefore, the differentiation of the mandibular condylar cartilage stopped at the preosteoblast (skeletoblast) stage. The lateral pterygoid muscle was attached to this anlage, and relatively abundant mesenchymal condensations were also formed at the muscle-attaching sites, e.g. the anlage of the mandibular body, the angular and coronoid processes. Three-dimensional reconstruction models showed that each mesenchymal condensation was connected to one another, and roughly outlined the shape of the mandible. Meckel's cartilage in the Runx2-deficient mice had two ectopic cartilaginous processes to which the digastric and myohyoid muscles were attached. These findings indicate that Runx2 is essential for the formation of the mandibular condylar cartilage, as well as for normal development of Meckel's cartilage and that muscle tissues influence mandible morphology.

Lipid peroxidation products have been known to induce cellular adaptive responses and enhance tolerance against subsequent oxidative stress through up-regulation of antioxidant compounds and enzymes. 24S-hydroxycholesterol (24SOHC) which is endogenously produced oxysterol in the brain plays an important role in maintaining brain cholesterol homeostasis. In this study, we evaluated adaptive responses induced by brain-specific oxysterol 24SOHC in human neuroblastoma SH-SY5Y cells. Cells treated with 24SOHC at sub-lethal concentrations showed significant reduction in cell death induced by subsequent treatment with 7-ketocholesterol (7KC) in both undifferentiated and retinoic acid-differentiated SH-SY5Y cells. These adaptive responses were also induced by other oxysterols such as 25-hydroxycholesterol and 27-hydroxycholesterol which are known to be ligands of liver X receptor (LXR). Co-treatment of 24SOHC with 9-cis retinoic acid, a retinoid X receptor ligand, enhanced the adaptive responses. Knockdown of LXRβ by siRNA diminished the adaptive responses induced by 24SOHC almost completely. The treatment with 24SOHC induced the expression of LXR target genes, such as ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1). The 24SOHC-induced adaptive responses were significantly attenuated by siRNA for ABCG1 but not by siRNA for ABCA1. Taken together, these results strongly suggest that 24SOHC at sub-lethal concentrations induces adaptive responses via transcriptional activation of LXR signaling pathway, thereby protecting neuronal cells from subsequent 7KC-induced cytotoxicity.

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