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Cell density dependent regulation of AP‐1 activity is important for chondrogenic differentiation of C3H10T1/2 mesenchymal cells
Author(s) -
Seghatoleslami M. Reza,
Tuan Rocky S.
Publication year - 2001
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.10019
Subject(s) - chondrogenesis , microbiology and biotechnology , ap 1 transcription factor , mesenchymal stem cell , chemistry , cellular differentiation , cell culture , transcription factor , biology , biochemistry , gene , genetics
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. J. Cell. Biochem. 84: 237–248, 2002. © 2001 Wiley‐Liss, Inc.