Positive and negative regulation of chondrogenesis by splice variants of PEBP2?A/CBF?1 in clonal mouse EC cells, ATDC5

Abstract

The αA type of the α subunit of the polyomavirus enhancer binding protein 2 (PEBP2αA), also called the core binding factor α1 (CBFα1) or til-1, plays crucial roles in osteogenesis. Little is known, however, about the function of PEBP2αA in chondrogenesis. Here, we examined the role of PEBP2αA in chondrogenesis of clonal mouse embryonal carcinoma cells, ATDC5, which are committed as chondroprogenitors. We found that as ATDC5 cells condensed and formed cartilaginous nodules, PEBP2αA increased, and the level was maintained throughout the process of chondrocytic maturation. When an established dominant negative form of PEBP2αA was introduced in undifferentiated ATDC5 cells, the cellular condensation and the subsequent processes were inhibited. This inhibition was overcome with BMP-4 treatment, which increased the endogenous expression of PEBP2αA. Thus, the process of chondrogenesis is regulated by the level of PEBP2αA activity. Along with the wild-type PEBP2αA, a splice variant form, til-1 G2, is naturally expressed in ATDC5 cells. In luciferase reporter assays, til-1 G2 not only exhibited a limited ability to transactivate the osteocalcin promoter but also inhibited the activity achieved by the wild-type PEBP2αA. When til-1 G2 was overexpressed by stable transfection in undifferentiated ATDC5 cells, it inhibited the progression of chondrogenesis. Therefore, we conclude that PEBP2αA acts as a positive regulator of chondrogenesis, and that this positive effect may be finely tuned by the opposing effect of the til-1 G2 isoform