Tissue Stiffening Promotes Keratinocyte Proliferation Through Activation of Epidermal Growth Factor Signaling Fiona N
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© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780 RESEARCH ARTICLE Tissue stiffening promotes keratinocyte proliferation through activation of epidermal growth factor signaling Fiona N. Kenny1, Zoe Drymoussi1, Robin Delaine-Smith2,3, Alexander P. Kao2, Ana C. Laly1,3, Martin M. Knight2,3, Michael P. Philpott1 and John T. Connelly1,3,* ABSTRACT The EGF receptor (EGFR) is a receptor tyrosine kinase that is highly Tissue biomechanics regulate a wide range of cellular functions, but expressed in the basal layer of the epidermis and, upon binding of the influences on epidermal homeostasis and repair remain unclear. EGF ligands, such as EGF, amphiregulin and transforming growth α α Here, we examined the role of extracellular matrix stiffness on human factor (TGF- ), the receptor dimerizes and becomes activated by keratinocyte behavior using elastomeric substrates with defined autophosphorylation at multiple tyrosine (Y) residues (Jost et al., mechanical properties. Increased matrix stiffness beyond normal 2000). Under homeostatic conditions, EGF signaling promotes physiologic levels promoted keratinocyte proliferation but did not alter growth and survival of basal keratinocytes through downstream the ability to self-renew or terminally differentiate. Activation of activation of mitogen activated protein kinase (MAPK) and epidermal growth factor (EGF) signaling mediated the proliferative phosphotidylinositide 3-kinase (PI3K) signaling pathways (Assefa response to matrix stiffness and depended on focal adhesion et al., 1997; Wan et al., 2001). However, overexpression of EGFR or assembly and cytoskeletal tension. Comparison of normal skin with its ligands is associated with a variety of hyperproliferative keloid scar tissue further revealed an upregulation of EGF signaling conditions, such as psoriasis (Piepkorn, 1996) and cancer (Reiss within the epidermis of stiffened scar tissue. We conclude that matrix and Sartorelli, 1987; Uribe and Gonzalez, 2011). stiffness regulates keratinocyte proliferation independently of changes While the roles of many biochemical factors in the regulation of in cell fate and is mediated by EGF signaling. These findings provide keratinocyte function have been described in detail, little is known mechanistic insights into how keratinocytes sense and respond to their about the contribution of mechanical or biophysical cues. In our mechanical environment, and suggest that matrix biomechanics may previous studies, we used micro-patterned substrates and established play a role in the pathogenesis keloid scar formation. that simple changes in keratinocyte shape and adhesion are potent regulators of terminal differentiation (Connelly et al., 2010). KEY WORDS: Mechanotransduction, Keratinocyte, Epidermis, EGF, Similarly, reduced tethering of ECM molecules to cell culture Keloid, Proliferation supports can induce terminal differentiation (Trappmann et al., 2011). While bulk material stiffness appears to have little effect on INTRODUCTION keratinocyte differentiation, the impact on additional cell functions In the epidermis of the skin, the balance between keratinocyte or fate over longer time scales has yet to be determined. As tissue proliferation in the basal layer and terminal differentiation and stiffness regulates the proliferation and self-renewal of multiple cell shedding in the upper layers maintains normal tissue homeostasis types, including mammary epithelia (Klein et al., 2009; Paszek et al., (Blanpain and Fuchs, 2009). These processes depend on a variety of 2005), muscle-derived stem cells (Gilbert et al., 2010), hematopoietic extracellular cues and signals, such as soluble growth factors (Reiss stem cells (Lee-Thedieck et al., 2012) and mesenchymal stem cells and Sartorelli, 1987; Rheinwald and Green, 1977; Zhu and Watt, (Chowdhury et al., 2010), it may also be an important mediator of 1999), cell-cell adhesion (Green and Simpson, 2007; Niessen, 2007), epidermal keratinocyte growth. and cell-extracellular matrix (ECM) interactions (Adams and Watt, In the present study we investigated the effects of altered matrix 1989; Jones and Watt, 1993). Dysregulation of key extrinsic signaling stiffness on keratinocyte behavior using model silicone substrates. pathways can lead to an imbalance in growth and differentiation and We show that increased matrix stiffness promotes epidermal often contributes to the pathogenesis of skin diseases including proliferation independently of changes in cell fate, and that EGF chronic wounds (Herrick et al., 1992; Stojadinovic et al., 2005; signaling mediates this response. We also demonstrate that EGF Wysocki et al., 1993), blistering (Bruckner-Tuderman et al., 1989), signaling is elevated within keloid scar tissue, which is ∼30-fold and cancer progression (Gat et al., 1998; Martins et al., 2009; Reiss stiffer than normal skin. These findings provide significant insights and Sartorelli, 1987; Uribe and Gonzalez, 2011). into the mechanisms of mechanosensing within the epidermis, and Epidermal growth factor (EGF) signaling is one of the major their impact on tissue homeostasis and scar formation. regulatory axes controlling keratinocyte proliferation and survival. RESULTS 1Centre for Cell Biology and Cutaneous Research, Barts and the London School of Substrate stiffness regulates keratinocyte proliferation Medicine and Dentistry, Queen Mary University of London, London E1 2AT, UK. independently of cell fate 2School of Engineering and Materials Science, Queen Mary University of London, To investigate the influence of matrix stiffness on long-term London E1 4NS, UK. 3Institute of Bioengineering, Queen Mary University of London, London E1 4NS, UK. keratinocyte growth and differentiation, we generated cell culture substrates with defined elastic moduli using polydimethylsiloxane *Author for correspondence ( [email protected]) (PDMS). PDMS substrates were crosslinked with 2% or 20% J.T.C., 0000-0002-5955-8848 (w/w) curing agent to produce non-porous substrates with elastic moduli of 180 kPa or 2 MPa, respectively (Fig. S1). Our previous Received 22 January 2018; Accepted 11 April 2018 atomic force microscopy (AFM) analysis of normal skin measured Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780 the elastic modulus of the basement membrane to be ∼140 kPa To assess whether substrate stiffness influenced epidermal cell (Kao et al., 2016). Thus, the PDMS substrates with 2% curing fate, keratinocytes were first expanded clonally on 2% or 20% agent were most similar to normal skin, while the substrates PDMS for 10 days, then dissociated and expanded a second time on crosslinked with 20% curing agent represented a ten-fold increase 2% or 20% PDMS. Keratinocytes formed similar numbers of in matrix stiffness. colonies under all conditions (Fig. 1G,H), indicating that previous Primary human keratinocytes were seeded onto 2% or 20% exposure to a soft or stiff environment did not affect the proportion PDMS substrates at clonal density and cultured for 10 days in low- of colony-initiating cells within the culture, a common read-out of Ca2+, serum-free medium with 0.1 ng/ml EGF. Cells formed a epidermal stem cell function in vitro (Jones and Watt, 1993). In similar number of colonies on both substrates but the colonies on the addition, expression of the terminal differentiation marker stiff 20% substrates were significantly larger (Fig. 1A,C). Tracking involucrin, was similar for cells cultured on 2% or 20% PDMS 2+ of individual colonies over the first seven days revealed a more for 5 days followed by stimulation with Ca (1.8 mM CaCl2) for rapid, exponential increase in the number of cells per clone on the 2 days to induce terminal differentiation (Fig. 1I). Likewise, there 20% substrates compared to the 2% substrates (Fig. 1D), and were no striking differences in Ca2+-induced assembly of adherens keratinocytes on the stiff substrates had a higher proliferative rate at junctions over this range of substrate moduli (Fig. S4). Taken day 7 (Fig. 1E,F). There were no detectable differences in initial together, these findings indicate that increased substrate stiffness adhesion or viability of keratinocyte cultured on the soft and stiff specifically stimulates keratinocyte proliferation but does not affect substrates (Fig. S2). adhesion, survival or terminal differentiation. We conclude that Fig. 1. Matrix stiffness regulates keratinocyte proliferation. (A) Representative images of colony formation by primary human keratinocytes cultured for 10 days on PDMS surfaces crosslinked with 2% or 20% curing agent and stained with Crystal Violet. (B,C) Quantification of colony number (B) and size (C) from scanned images of stained wells. Data represent mean±s.e.m. (n=4 experiments), *P<0.05. (D) Quantification of the average number of cells per colony on 2% and 20% PDMS based on bright-field images (10×) at defined locations, tracked from day 3–7. (E,F) Representative images and quantification of EdU-positive keratinocytes cultured on 2% (E) or 20% (F) collagen-coated PDMS for 7 days. Scale bars: 100 µm. Data represent mean±s.e.m. (n=4 experiments), *P<0.05. (G) Representative images of colony formation on 2% or 20% PDMS following an initial expansion on either 2% or 20% PDMS. (H) Quantification of colony number following expansion on 2% then 2% (2/2), 2% then 20% (2/20), 20% then 2% (20/2), or 20% then 20% (20/20). Data represent mean±s.e.m. (n=3 experiments), *P<0.05.