Two of four alternatively spliced isoforms of RUNX2 control osteocalcin gene expression in human osteoblast cells

• Published in: Gene 2008-04, Vol.413 (1), p.8-17
• Main Authors: Makita, Naoyuki, Suzuki, Mitsuhiro, Asami, Shiori, Takahata, Rintaroh, Kohzaki, Daika, Kobayashi, Sho, Hakamazuka, Takashi, Hozumi, Nobumichi
• Format: Article
• Language: English
• Subjects: Alternative Splicing; Base Sequence; Binding Sites - genetics; Cell Differentiation; Cell Line, Tumor; Cell Nucleus - metabolism; Cells, Cultured; Chondrocytes - cytology; Chondrocytes - metabolism; Chondrosarcoma - genetics; Chondrosarcoma - metabolism; Core Binding Factor Alpha 1 Subunit - genetics; Core Binding Factor Alpha 1 Subunit - metabolism; Cytosol - metabolism; DNA Primers - genetics; Gene Expression Regulation; Histone Deacetylases - metabolism; Humans; Mesenchymal Stromal Cells - cytology; Mesenchymal Stromal Cells - metabolism; Osteoblast; Osteoblasts - cytology; Osteoblasts - metabolism; Osteocalcin; Osteocalcin - genetics; Osteosarcoma - genetics; Osteosarcoma - metabolism; p300-CBP Transcription Factors - metabolism; Promoter Regions, Genetic; Protein Isoforms - genetics; Protein Isoforms - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RUNX2; Transcriptional regulation; Transfection
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2007.12.025

Runx2 is a Runt domain transcription factor that transcriptionally regulates osteoblast differentiation and bone formation. In this study, we show that human chondro- and osteosarcoma cell lines, human mesenchymal stem cells (hMSC) and a human primary chondrocytes (HC), osteoblst cells (HOb) express an intact isoform (RUNX2wt) and 3 alternatively spliced isoforms (RUNX2Δ5, Δ7, and Δ5Δ7) that are generated by skipping exon 5 and/or exon 7. Two of the truncated forms of RUNX2 (RUNX2Δ5 and RUNX2Δ5Δ7) did not localize in the nucleus and had lost their DNA binding activity. In cotransfection experiments with an osteocalcin ( OC) promoter construct, we confirmed that only RUNX2wt and RUNX2Δ7 could upregulate the OC promoter activity in the osteosarcoma cell line. In addition, the coactivator CBP/p300 enhanced the transcriptional activity of the OC promoter when coexpressed with RUNX2wt or RUNX2Δ7, but not when coexpressed with RUNX2Δ5 or RUNX2Δ5Δ7. In contrast, the corepressor HDAC3 only repressed the activation from the OC promoter when coexpressed with RUNX2wt. These results support the hypothesis that RUNX2 both up- and downregulates its target gene promoters, as exemplified by the OC gene, using various isoforms and context-dependent formation of transcriptional complexes.