Cell density dependent regulation of AP-1 activity is important for chondrogenic differentiation of C3H10T1/2 mesenchymal cells.

Abstract

The multipotential C3H10T1/2 mesenchymal cells undergo chondrogenic differentiation only when seeded as high-density micromass cultures, particularly upon treatment with bone morphogenetic protein-2 (BMP-2). The molecular mechanism(s) responsible for the cell density-dependent onset of cartilage-specific gene expression is presently unknown. Interestingly, a number of recent studies have indicated that activating protein-1 (AP-1), a well known downstream target of the mitogenic activated protein kinase (MAP kinase) signaling pathway, is a target of chondrogenic/osteogenic growth factors such as BMP-2, and plays a role in osteogenic gene regulation as well as in chondrogenic differentiation. The aim of this study is to examine the density-dependent alteration in the level and binding activity of AP-1 and its functional involvement in C3H10T1/2 mesenchymal chondrogenesis. To measure the activity of the AP-1 transcription factor, we generated a pool of stable C3H10T1/2 cell lines harboring a luciferase expression vector driven by a concatamer of an efficient AP-1 response element (AP1-10T1/2 cells). Luciferase activity of AP1-10T1/2 cultures was found to decrease sharply with increase in cell density, either as a function of culture time or initial cell seeding densities. In C3H10T1/2 micromass cultures undergoing chondrogenesis, AP-1 activity was further reduced and then maintained at a low, steady level for the entire 3-4 day culture period. AP-1 activity in micromass cultures was not significantly affected by BMP-2 treatment, but chondrogenesis was compromised upon competitive inhibition of AP-1 activity with a double-stranded AP-1 binding oligonucleotide. The level of AP-1 binding correlated with the activity of its response element but not with the levels of its leucine-zipper containing subunits, c-Jun and c-Fos. These findings suggest that a cell density-dependent, low but steady level of AP-1 binding and activity is required for promoting the chondrogenic potential of C3H10T1/2 cells.