Previous studies of EGFR-mediated effects on MSC osteogenic differentiation have produced diverse findings (Krampera et al., 2005
; Sibilia et al., 2003
; Tamama et al., 2006
), reflecting the intricate dynamics of EGF ligand/receptor system trafficking and signaling. Here, we studied MSC responses to EGFR stimulation using a mode of ligand presentation that offers a relatively constant exogenous EGF stimulus to the cells. Our experiments show that stimulation of primary human MSC with tEGF increases osteogenic differentiation markers ALP (at day 7) and mineralization (day 21). This phenotypic behavior is associated with sustained activation of the EGFR (, ) and Erk () and increased ratios of pEGFR/total EGFR for cells on tEGF compared to control. The physical tethering presumably constrains ligand/receptor complexes from undergoing endocytic internalization, protecting activated EGFR from a degradation fate associated with EGF-mediated internalization. Consistent with the concept that sustained EGFR activation in the day 4–7 period enhances osteogenic differentation, both EGFR downregulation by soluble EGF (sEGF) and inhibition of EGFR kinase activity by a pharmacological agent each independently served to attenuate tEGF-induced increase in alkaline phosphatase expression (). The delay in EGFR (and HER2) expression level increase on tEGF substrata following initial cell seeding () coupled with the concomitant greater EGFR phosphorylation level () indicates that it is the integrated combination of receptor expression and ligand availability during the first few days of osteoinduction that facilitates enhanced downstream signaling activities governing MSC differentiation fate decisions by day 7.
Our results taken together suggest the model shown schematically in : the maintenance of higher levels of pEGFR, due in part to inhibition of EGFR internalization and in part to sustained stimulation by externally-restrained EGF ligand, both contribute to enhancement of differentiation.
Conceptual illustration of tEGF effects in MSC osteogenic differentiation
The literature has conflicting reports regarding the effects of EGF on osteogenic differentiation of MSCs. 0.4 ng/ml EGF (66.4 pM) (Kumegawa et al., 1983
), 5–50 ng/ml (0.5–5 nM) of amphiregulin (Qin et al., 2005
), and 50 ng/ml (2.3 nM) HB-EGF, (Krampera et al., 2005
) all have been found to inhibit osteogenic differentiation. In contrast, addition of 50 ng/ml (83 nM) EGF with media changes every 4 days (Kratchmarova et al., 2005
) and 5 ng/ml EGF (8.3 nM) (Kim et al., 2008
) were observed to increase osteogenic differentiation. Different EGF concentrations, treatment durations, cross-binding to other EGFR family members (by amphiregulin and HB-EGF), and cell culture histories render these reports difficult to interpret as a whole, particularly since the different bone progenitor cell types used for these experiments have different levels of EGFR (Tamama et al., 2006
We have moreover found differential regulation of EGF and TGFα synthesis (), offering an additional complicating feature in understanding EGFR-mediated regulation of MSC differentiation but one that is consistent with our notion of the central importance of time-integrated combination of receptor level and ligand availability, as these two ligands can have very different consequences for receptor and ligand endocytic degradation. In fibroblasts, EGF binding predominantly traffics ligand/receptor complexes to lysosomes for degradation when receptor expression is relatively low, whereas TGFα bindings instead favors trafficking to recycling (French et al., 1995
; Wiley, 2003
). We observe here that inhibition of EGFR kinase activity yields an increase of TGFα mRNA () with greater fold increase than of EGF mRNA (). We speculate that this differential regulation in cells with depressed EGFR signaling provides a mechanism for the cells to sustain EGFR signaling via TGFα by sparing EGFR number in the longer-term (Joslin et al., 2007
Future work will be required to elucidate the complex relationships between particular signals – including magnitude and duration – and MSC differentiation fate downstream of EGFR activity. ERK signaling has been implicated in osteogenesis (Ge et al., 2007
; Jaiswal et al., 2000
), and tuning of the ERK signaling pathway with inhibitors promoted differentiation of MSCs and preosteoblasts (Higuchi et al., 2002
). Thus, modulating this key pathway with a sustained stimulus may help promote osteogenic differentiation analogously. In fact, our highly controllable tethered ligand system may offer prospects for generating novel, beneficial signaling network dynamics. It is possible that tEGF-generated sustained activation and more continuously engaged downstream pathway components (such as ERK) elicits enhancement of phosphatase-related feedback loops, with subsequent muting of cell response to other exogenous signals. Such desensitization has been shown in NIH-3T3 cells: adhesion to extracellular matrices activated ERK intracellular signaling but desensitized the cell to later growth factor stimulation and responsiveness (Galownia et al., 2007
). Full activation of ERK and other mitogen-activated kinases (MAPKs) typically develops only following normal endocytic trafficking with hyperphosphorylation of EGFR in the endosomal compartment (Vieira et al., 1996
). Receptors constrained at the cell plasma membrane by tethered ligand would not be expected to activate pathways preferentially localized to endosomal compartments.
Restricting EGFR to the plasma membrane via tEGF may also preferentially activate PI3K/Akt paths (Watton and Downward, 1999
; Wu et al., 2000
). Although inhibition of the PI3K pathway in immortalized MSCs has been determined to increase osteogenic differentiation (Kratchmarova et al., 2005
), their protocol of stimulation with a large bolus of sEGF may have activated a different network and effectors, and other groups have shown that inhibition of PI3K decreases osteogenic differentiation (Kundu et al., 2008
The phenotypic outcomes we measured here, ALP and matrix mineralization, are influenced by activation of osteoblastic transcription factors Runx2 and Osterix. Future studies directed at illuminating how the EGFR pathways influence MSC fate choices may productively include these features of the network, as motivated by our results here. This work shows that tethering strategies that alter receptor dynamics and signaling, can, ultimately, be used to modify MSC behavior in ways that may be useful for regenerative medicine strategies.