Collective processes such as wound re-epithelialization result from the integration of individual cellular decisions. To determine which individual cell behaviors represent the most promising targets to engineer re-epithelialization, we examined collective and individual responses of HaCaT keratinocytes seeded upon polyacrylamide gels of three stiffnesses (1, 30, and 100 kPa) and treated with a range of epidermal growth factor (EGF) doses. Wound closure was found to increase with substrate stiffness, but was responsive to EGF treatment only above a stiffness threshold. Individual cell behaviors were used to create a partial least squares regression model to predict the hierarchy of factors driving wound closure. Unexpectedly, cell area and persistence were found to have the strongest correlation to the observed differences in wound closure. Meanwhile, the model predicted a relatively weak correlation between wound closure with proliferation, and the unexpectedly minor input from proliferation was successfully tested with inhibition by aphidicolin. Combined, these results suggest that the poor clinical results for growth factor-based therapies for chronic wounds may result from a disconnect between the individual cellular behaviors targeted in these approaches and the resulting collective response. Additionally, the stiffness-dependency of EGF sensitivity suggests that therapies matched to microenvironmental characteristics will be more efficacious.
Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.
BackgroundThe insulin-like growth factor (IGF) system impacts cellular development by regulating proliferation, differentiation, and apoptosis, and is an attractive therapeutic target in cancer. The IGF system is complex, with two ligands (IGF1, IGF2), two receptors (IGF1R, IGF2R), and at least six high affinity IGF-binding proteins (IGFBPs) that regulate IGF ligand bioavailability. While the individual components of the IGF system are well studied, the question of how these different components integrate as a system to regulate cell behavior is less clear.ResultsTo analyze the relative importance of different mechanisms that control IGF network activity, we developed a mass-action kinetic model incorporating cell surface binding, phosphorylation, and intracellular trafficking events. The model was calibrated and validated using experimental data collected from OVCAR5, an immortalized ovarian cancer cell line. We then performed model analysis to examine the ability of IGF2R or IGFBPs to counteract phosphorylation of IGF1R, a critical step for IGF network activation. This analysis suggested that IGF2R levels would need to be 320-fold greater than IGF1R in order to decrease pIGF1R by 25 %, while IGFBP levels would need to be 390-fold greater. Analysis of The Cancer Genome Atlas (TCGA) data set suggested that this level of overexpression is unlikely for IGF2R in ovarian, breast, and colon cancer. In contrast, IGFBPs can likely reach these levels, suggesting that IGFBPs are the more critical regulator of IGF1R network activity. Levels of phosphorylated IGF1R were insensitive to changes in parameters regulating the IGF2R arm of the network.ConclusionsUsing a mass-action kinetic model, we determined that IGF2R plays a minor role in regulating the activity of IGF1R under a variety of conditions and that due to their high expression levels, IGFBPs are the dominant mechanism to regulating IGF network activation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0263-6) contains supplementary material, which is available to authorized users.
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