A novel mechanism for the regulation of osteoblast differentiation: Transcription of periostin, a member of the fasciclin I family, is regulated by the bHLH transcription factor, twist

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Abstract
Periostin is a secreted protein that is highly expressed in early osteoblastic cells in vitro and in periosteum and periodontal ligament tissues in vivo. It is known that periostin supports cellular adhesion and spreading in vitro. Although, the mechanisms of transcriptional regulation of periostin are poorly understood, gene‐profiling data have revealed that overexpression of Twist, a basic helix‐loop‐helix (bHLH) transcription factor, resulted in increased periostin expression as validated by Northern blot and reverse transcription‐polymerase chain reaction (RT‐PCR) analyses. Twist is an important transcription factor for cell type determination and differentiation and has been shown to play an important regulatory role in early osteogenesis. In situ hybridization of mouse calvarial bones indicated that periostin and Twist mRNA are co‐localized at the osteogenic fronts of calvarial bones. To characterize the 5′ flanking region of the periostin gene, primer extension was carried out to identify the transcription start site, and DNA sequence analysis confirmed the presence of a ‘Twist‐box’ response element. The results of electrophoretic mobility shift assay (EMSA) using nuclear extracts of MC3T3‐E1 cells revealed that Twist bound to the Twist‐box sequence on the periostin promoter. In vivo footprinting experiments using ligation‐mediated PCR (LM‐PCR) indicated that the Twist‐box sequence was protected in undifferentiated MC3T3‐E1 preosteoblasts but not in differentiated MC3T3‐E1 osteoblasts. To determine whether Twist actually regulates the periostin expression, 293T cells were transiently co‐transfected with the periostin promoter construct and the human Twist expression vector. Reporter analysis indicated that the periostin promoter activities were enhanced by overexpression of Twist. These data suggest that Twist can bind to the periostin promoter in undifferentiated preosteoblasts and up‐regulate periostin expression, consistent with the up‐regulation of periostin expression by Twist as observed in the gene‐profiling data. J. Cell. Biochem. 86: 792–804, 2002.