ABSTRACT
The nuclear receptor, ultraspiracle protein (USP), is a transcription factor and an essential component of a heterodimeric receptor complex with ecdysone receptor. However, the mechanisms underlying the transcriptional regulation of USP in silkworm are unknown. In this study, using dual-spike-in qPCR method, we examined the expression of Bombyx ultraspiracle gene (BmUSP) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. The results showed that the expression levels of BmUSP gene varied in different tissues and were increased 2 h after exposure to ecdysone. To identify the molecular mechanism underlying the regulation of USP gene expression in silkworm Bombyx mori, promoter truncation analyses were performed using the luciferase reporter assay and Bac-to-Bac expression system in several tissues of B. mori. BmUSP gene promoter with 5' end serial deletions showed different levels of activity in various tissues, higher in fat body and Malpighian tubule. Deletion of the region from -485 to -445 and -307 to -281 upstream of BmUSP gene abolished and increased its promoter activity, respectively. This region contains AP-1, Dfd transcription factor binding sites. These results indicate that BmUSP are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. This study would provide an important foundation for investigating the mechanism underlying the transcriptional regulation of BmUSP in the silkworm.
Subject(s)
Bombyx/genetics , Gene Expression Regulation , Insect Proteins/genetics , Promoter Regions, Genetic , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , Animals , Bombyx/metabolism , Ecdysone/pharmacology , Insect Proteins/metabolism , Organ Specificity , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolismABSTRACT
To study and evaluate BMP7s functions in osteogenic differentiation of human periosteal cells in vitro. Human periosteal cells from adult tibia were collected and cultured as experimental samples. BMP7 was used to induce periosteal cells in the experiment group with common osteogenic medium. The proliferative activity of periosteal cells was detected by CCK-8. The potentials of osteogenic differentiation were demonstrated as follows: (1) realtime-PCR and ELISA to confirm the expression of the OC, ALP and OPN, (2) Colorimetry, ALP staining and Von Kossa staining were performed to identify ALP activity, ALP expression and calcium nodules, respectively. Based on the significant different expression of OC, ALP and OPN, BMP7 ability of osteogenic differentiation can be identified. ALP activity detection, calcium nodules staining and toluidine staining also provide the power evidence to support BMP7 can promote osteogenic differentiation of human periosteal cells in vitro. To human periosteal cells, BMP7 is a good inducer for osteogenic differentiation. Therefore, it's maybe a potential tool for clinical application.
