The role of Sp1 in BMP2-up-regulated Erk2 gene expression

Abstract

Extracellular signal-regulated kinase (Erk) is an important component in many cellular processes, including cell differentiation and proliferation. We previously showed that Erk is involved in BMP2-induced osteoblastic differentiation in mesenchymal progenitor cells and Erk protein level is up-regulated under BMP2 inducement. In this study, the molecular mechanism which mediates the regulation of Erk2 gene expression by BMP2 was investigated. Northern blot analysis showed that increased Erk2 protein level under BMP2 inducement comes from BMP2-up-regulated Erk2 mRNA expression. Transient transfection of C3H10T1/2 cells with a series of constructs of mouse Erk2 promoter demonstrated that a sequence residing between nucleotides −148 and −42 of Erk2 promoter is one of the BMP2-responsive elements. Electrophoresis mobility shift assays indicated that BMP2 treatment on C3H10T1/2 cells increases the binding of cell nuclear extracts to the −148/−42 fragment, and the BMP2-enhanced binding bands are Sp1 transcription factors. A series of competitive gel shift assays and the supershift assays by mapping oligos S1–S5 on −148/−42 identified that S1 and S5 contain Sp1 binding sites, which are located, respectively, in −147/−139 and −51/−46. Transfection studies showed that the addition of the Sp1 binding inhibitor mithramycin or mutation of the Sp1 site residing at −147/−139 abolishes the up-regulation of Erk2 promoter activity induced by BMP2. All these results indicate that Sp1-mediated transcription is one of the mechanisms, which is responsible for BMP2-induced up-regulation of Erk2 expression.