Sustained epidermal growth factor receptor levels and activation by tethered ligand binding enhances osteogenic differentiation of multi-potent marrow stromal cells.
Epidermal growth factor receptor (EGFR)-mediated signaling helps regulate bone development and healing through its effects on osteogenic cells. Here, we show how EGFR activity and osteogenic differentiation responses in primary human bone marrow-derived multipotent stromal cells (MSCs) are influenced by presenting covalently tethered epidermal growth factor (tEGF) on the culture substratum, a presentation mode that reduces EGFR internalization and restricts signaling to the cell surface. In both absence and presence of tEGF, MSCs increase expression levels of EGFR and its heterodimerization partner HER2 during the course of osteogenic differentiation. tEGF substrata increased levels of phosphorylated EGFR and phosphorylated extracellular regulated kinase (ERK) compared to control substrata, and these elevations were associated with a twofold enhancement of MSC alkaline phosphatase activity at day 7 and matrix mineralization at day 21. Surprisingly, addition of soluble EGF (sEGF) to cells cultured on tEGF substrata reduces osteogenic differentiation, even though EGFR signaling is more strongly activated in acute, short-term manner by sEGF treatment than by tEGF treatment. A striking concomitant result of the sEGF effects is near-complete downregulation of EGFR and HER2, demonstrating that the tEGF/EGFR interaction is dynamically reversible even though temporally sustained. Taken together, our results show that enhanced MSC osteogenic differentiation corresponds to a sustained combination of receptor expression and ligand presentation, both of which are maintained by tEGF.
Platt
Manu O
MO
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Roman
Arian J
AJ
Wells
Alan
A
Lauffenburger
Douglas A
DA
Griffith
Linda G
LG
D001854:Bone Marrow Cells / Q000166:cytology* / Q000201:enzymology; D001857:Bone Matrix / Q000378:metabolism; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000201:enzymology; D010012:Osteogenesis; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D017154:Stromal Cells / Q000166:cytology* / Q000201:enzymology; D013997:Time Factors; D016211:Transforming Growth Factor alpha / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D012333:RNA, Messenger; D016211:Transforming Growth Factor alpha; D004815:Epidermal Growth Factor; D066246:ErbB Receptors