Abstract
Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.
Highlights
Chondromodulin-I (ChM-I)1 is a 25-kDa glycoprotein originally purified from bovine epiphyseal cartilage on the basis of growth-promoting activity for chondrocytes [1] and subsequently revealed to be a potent vascular endothelial cell growth inhibitor [2]
To confirm that the expression of cartilage-related genes was derived from tumor cells and not from surrounding normal tissues, the expression of the same set of genes was investigated in seven OS cell lines (Fig. 1B)
The expression of the SOX9 gene was observed in all of the cell lines including MG63 and Saos2 in which none of cartilage-related genes other than the SOX9 was expressed. These data indicated that the expression of the cartilage-related genes in OS stemmed from tumor cells and that some OS tumor cells expressed bonerelated genes as well as cartilage-related genes of which the expression was regulated by some mechanisms other than the expression of the SOX9 gene
Summary
ChM-I, chondromodulin-I; OS, osteosarcoma; OBOS, osteoblastic OS; FBOS, fibroblastic OS; CBOS, chondroblastic OS; 5-Aza-dC, 5-aza-2-deoxycytidine; RT, reverse transcription; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; AGC, aggrecan. The expression of the ChM-I gene is limited to cells in the resting, proliferating, and early hypertrophic zone of the growth plate [2,3,4] These results suggest that the expression of the ChM-I gene is regulated strictly in a cell- and stage-related manner, the molecular mechanisms leading to this spatiotemporal expression have not been elucidated. A particular intriguing subtype of OS is chondroblastic OS (CBOS) in which tumor cells directly produce immature cartilage in addition to osteoid [5, 9], suggesting that tumor cells in this subtype have the potential to differentiate into both osteogenic and chondrogenic cells These clinical findings suggest that the precursor cells of OS range from mesenchymal stem cells to mature osteoblasts and that OS cells can be used as materials to investigate the regulatory mechanisms of cell- and stage-specific genes such as the ChM-I gene. We found that the expression of the ChM-I gene was regulated positively by a transcription factor, Sp3, and that the binding of Sp3 was regulated by the methylation status in the core-promoter region of the ChM-I gene, especially at one Sp3 binding site
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