RESUMO
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 (RUNX2Delta5, Delta7, and Delta5Delta7) that are generated by skipping exon 5 and/or exon 7. Two of the truncated forms of RUNX2 (RUNX2Delta5 and RUNX2Delta5Delta7) 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 RUNX2Delta7 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 RUNX2Delta7, but not when coexpressed with RUNX2Delta5 or RUNX2Delta5Delta7. 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.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Processamento Alternativo , Sequência de Bases , Sítios de Ligação/genética , Diferenciação Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Condrossarcoma/genética , Condrossarcoma/metabolismo , Citosol/metabolismo , Primers do DNA/genética , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
We have synthesized eight novel 16-ene-22-thia-26,27-dimethyl-19-norvitamin D3 analogs 1-5 bearing side chains of different sizes, in combination with 20S- and 20R-isomers. The target compounds were prepared by Wittig-Horner reaction of A-ring phosphine oxide with 16-ene-22-thia-25-hydroxy Grundmann's ketones having different sized side chains, which were derived from the S-phenyloxycarbonyl derivative 13 as key intermediates. The binding affinity to the vitamin D receptor (VDR), VDR-mediated transcriptional activity, and osteoclast-inducing activity of synthetic 22-thia-19-norvitamin D analogs 1-5 were investigated. The (20S)-22-thia-19,24-dinorvitamin D analog 1a is as active as the natural hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) in terms of biological activities tested in vitro. The analogs 2a and 3a exhibited almost the same potency as 1alpha,25-(OH)2D3 in binding to the VDR, were about 20 times more potent than 1alpha,25-(OH)2D3 in terms of transcriptional activity, and 3a was approximately 100 times as potent as 1alpha,25-(OH)2D3 in eliciting osteoclast formation. The biological activities of (20S)-22-thia compounds were more potent (by more than 10-fold) than those of the corresponding 20R-counterparts, but the activity of (20R)-compounds 1b, 2b, and 3b in stimulating the formation of osteoclasts was similar to that of 1alpha,25-(OH)2D3, and the 24-dihomo- and trihomo-analogs 4a and 5a showed low transcriptional activity. These results suggest that elongation of the side chain in 22-thia analogs by up to one carbon can be stably accommodated in the VDR ligand binding pocket.