p38 MAP Kinase Regulation of AP-2 Binding in TGF-?1-Stimulated Chondrogenesis of Human Trabecular Bone-Derived Cells
Collagenase-treated, explanted human trabecular-bone chips are an excellent source of osteoblast-like cells. We have recently shown the multiple differentiation potential of these cells; in addition to osteogenesis and adipogenesis, these cells also undergo chondrogenesis when maintained as high-density pellet cultures (250,000 cells/pellet) in a serum-free, chemically defined medium stimulated with TGF-?1 (10 ng/mL). In this investigation, we have analyzed how transactivating nuclear transcription factors, specifically AP-2 and SP-1, may interact with common cis-acting elements found in the regulatory region of cartilage-specific genes as part of the signal transduction mechanism of TGF-?1 and p38 during chondrogenesis of human trabecular bone-derived multipotential cells. Both TGF-?1 stimulation and p38 MAP kinase activation affect the binding of AP-2 as well as SP-1 to oligonucleotides with sequence similarity to the overlapping AP-2/SP-1 sites found in the putative 52-bp immediate upstream regulatory region and the 5?-untranslated region of the human aggrecan gene. Electrophoretic mobility shift assays show that TGF-?1 treatment of the bone-derived cells inhibits AP-2 DNA binding but enhances the DNA binding ability of SP-1. Additionally, treatment of these TGF-?1-stimulated cells with p38 MAP kinase inhibitor, SB203580, rescued the AP-2 DNA binding but did not affect SP-1 DNA binding. These findings indicate that AP-2 DNA binding is the target of both TGF-?1 and p38 MAP kinase signaling pathways and suggest a possible signal transduction cascade whereby TGF-?1 induction of chondrogenesis involves the activation of p38 MAP kinase and the subsequent inhibition of DNA binding by AP-2, thereby preventing the transcriptional repression of the aggrecan gene.
chondrogenesis mesenchymal progenitor cell human trabecular bone p38 mitogen-activated protein (map) kinase activated protein-2 (ap-2)