Regulation of keratinocyte signaling and function via changes in epidermal growth factor presentation

Abstract

Motivated by the need for bioactive materials that can accelerate dermal wound healing, this work describes the response of keratinocytes to covalently immobilized epidermal growth factor (EGF) and how differences in the physical presentation of this growth factor impact cell function. Specifically, human keratinocytes were cultured with EGF delivered in soluble form, immobilized in a homogeneous pattern, or immobilized in a gradient pattern, followed by analysis of cellular signaling, proliferation, and migration. By changing the manner in which EGF was presented, keratinocyte behavior was dramatically altered. Keratinocytes responded to immobilized EGF patterns with high EGF receptor (EGFR) but low ERK 1/2 and Akt phosphorylation, accompanied by low proliferation, high migratory activity, and coordinated cell alignment. In contrast, keratinocytes treated with soluble EGF experienced lower EGFR but higher ERK 1/2 and Akt phosphorylation, and displayed a highly proliferative, rather than migratory, phenotype. Keratinocytes also responded to differences in immobilized EGF patterns, as migration was fastest upon immobilized gradients of EGF. Better understanding the interaction of cells with soluble vs. immobilized growth factors can help elucidate native healing events and achieve greater control over cell function, which may be useful in the development of wound repair treatments for many types of tissues.

Covalent Growth Factor Immobilization Strategies for Tissue Repair and Regeneration

“…Several studies have found that immobilization of a growth factor does not just extend its lifespan, but can also change the manner in which that growth factor impacts cell behavior. [23,59] In other words, the cellular response to growth factors can be regulated by the manner in which that growth factor is presented (i.e., soluble vs. immobilized). The consequence of this phenomenon is that the cellular response to an immobilized growth factor cannot always be predicted by our knowledge of the cellular response to that same growth factor in solution.…”

“…Alternatively, photo-chemistry can be combined with WSC techniques, as is the case with the azidophenylderivatized polyallylamine synthesized by Ito et al, [20,21] where the growth factor is rendered photoreactive following reaction of growth factor carboxylic acids with the polyallylamine using WSC chemistry. These techniques, and minor variations thereof, have been used to covalently attach EGF to polystyrene [18,[20][21][22][23][24][25] or NGF to polypyrrole. [26] Novel variations of these techniques have also been used to gain control over orientation while immobilizing VEGF on self-assembled monolayers [27] or EGF on glass.…”

“…The potential for ultraviolet-induced damage to the growth factor has Figure 2. General reaction scheme for modifying a growth factor with a photo-reactive phenyl azide group and subsequent immobilization on a biomaterial substrate (figure adapted from [23] ). Specifically, this figure depicts the reaction of the N-hydroxysuccinimide end of the heterobifunctional crosslinking molecule sulfo-SANPAH with a primary amine in a growth factor, followed covalent immobilization of the modified growth factor onto a substrate upon application of UV light to initiate reaction via the phenyl azide.…”

“…Dermal wound healing Polystyrene [18,22,23] PCL/PCL-PEG [14] PCL/gelatin [13] General mitogen Polystyrene [25] PMMA [99] Hepatocyte culture Glass [28,100] Cartilage repair Chitosan [64] Stem cell differentiation (neural) SAMs [90] Polystyrene [21] Stem cell differentiation (osteoblast) PMMA-g-PEG [91] Stem cell survival PMMA-g-PEG [59] Corneal repair PDMS [101,102] IGF-1 Myogenic differentiation Silicone [103] Dermal wound healing Polystyrene [22] Pancreatic stem cell differentiation Glass [62] LIF Maintenance of ESC pluripotency POMA [29] PET [104] Gelatin [88] NGF Stem cell differentiation (neural) PCL/PCL-PEG [17] Neurite extension Glass [61] pHEMA [54] Polypyrrole [26] PDGF-AA Stem cell differentiation (neural) Agarose [55] PDGF-BB Angiogenesis/Vascularization PEG [35] Demineralized bone [41] VEGF Angiogenesis/Vascularization PEG [19,33] Collagen [12,40,56] SAMs [27] PLGA [4] Gelatin [105] Stem cell differentiation (vascular/blood) Chitosan/collagen IV [10] Titanium [52] Agarose [39] a) Polymer abbreviations: PCL, poly(caprolactone); PDMS, poly(dimethyl siloxane); PEG, poly(ethylene glycol); PET, poly(ethylene terephthalate); pHEMA, poly(hydroxyethyl m...…”

The Roles of Growth Factors in Keratinocyte Migration

“…[33][34][35] Interestingly, the mode in which the EGF ligand is presented to KCs also appears to be important for determining whether the cells migrate or proliferate; EGF on a stationary substrate induces KC migration, while soluble EGF induces KC proliferation, suggesting that soluble and membrane-or ECM-bound EGF-family ligands play distinct roles in directing KC activities. 36 Insulin growth factor family. Both insulin and IGF-1 bind to the insulin receptor (IR) and IGF-1 receptor (IGF1R) RTKs, with insulin binding with a higher affinity to IR than IGF1R and IGF-1 binding with a higher affinity to IGF1R than IR.…”