Functional analysis of pig myostatin gene promoter with some adipogenesis- and myogenesis-related factors.

Myostatin (MSTN) is primarily expressed in muscle and plays an important role in muscle and fat development in pigs. However, there is little information about the regulation of pig MSTN. In order to elucidate whether pig MSTN could be regulated by muscle- and fat-related factors, the porcine MSTN promoter was amplified and cloned into pGL3-basic vector, and transfected into cells to analyze the transcriptional activity of promoter with muscle- and fat-related factors through dual-luciferase reporter assays. 5'-deletion expression showed that there was a negative-regulatory region located between nucleotides -1519 and -1236 bp, and there were some positive-regulatory regions located between -1236 and -568 bp. The longest fragment (1.7 kb) was cotransfected with muscle-related transcription factor myogenic differentiation 1 (MyoD), resulting in promoter transcriptional activity upregulation. The fragment was treated by the adipogenic agents (DIM) including dexamethasone, insulin, and isobutyl-1-methylxanthine (IBMX). We found that MSTN promoter transcriptional activity can be regulated by IBMX, but not by DIM. CCAAT/enhancer binding protein (C/EBP) α and C/EBPβ, two proteins which are induced by DIM during adipogenesis were cotransfected with the 1.7-kb fragment, respectively, resulting in promoter transcriptional activity downregulation. Treating the fragment with rosiglitazone which induce the expression of peroxisome proliferator-activated receptor γ (PPARγ), resulting in promoter transcriptional activity upregulation. Cotransfection experiments confirmed this result. Taken together, we showed that porcine MSTN could be upregulated by IBMX, MyoD, and PPARγ but downregulated by C/EBPα and C/EBPβ.