sign in sign up
<<
Home , Keratinocyte

© 2018. Published by The Company of Biologists Ltd | Journal of Science (2018) 131, jcs215780. doi:10.1242/jcs.215780

RESEARCH ARTICLE Tissue stiffening promotes keratinocyte proliferation through activation of 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 and, upon binding of the influences on epidermal 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 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 with its ligands is associated with a variety of hyperproliferative keloid tissue further revealed an upregulation of EGF signaling conditions, such as (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 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 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 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 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 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 , 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. (I) Western blot analysis of involucrin expression in keratinocytes cultured on 2% or 20% collagen-coated PDMS for

5 days in KSFM followed by stimulation with 1.8 mM CaCl2 for 2 days. Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780 matrix stiffness regulates keratinocyte growth independently of immunofluorescence staining, while receptor internalization changes in cell fate. following treatment with EGF appeared to be unaffected by substrate stiffness (Fig. 2C). A similar increase in the basal level of Increased substrate stiffness activates EGF signaling phosphorylated Y845, Y1086 and Y1173 was also observed on the To gain insight into the mechanism by which matrix stiffness stiff substrates (Fig. 2D, Fig. S3A). regulates keratinocyte proliferation we first examined the effects on The effects of matrix stiffness on EGF signaling occurred in a EGF signaling, a key regulator of proliferation and survival (Jost dose-dependent manner. EGFR phosphorylation progressively et al., 2000). Cells were cultured on 2% or 20% PDMS substrates with increased with increasing substrate stiffness for PDMS crosslinked or without collagen coating for 24 h in EGF-free medium, then with 2%, 5%, 10% or 20% curing agent (Fig. 2D). There was also stimulated with 10 ng/ml EGF for 15 min. High levels of EGFR elevated EGFR phosphorylation in keratinocytes cultured on stiff phosphorylation at Y1068 were observed on all substrates following substrates coated with fibronectin; however, the response on EGF stimulation (Fig. 2A), but there was a significantly higher level fibronectin was associated with increased total EGFR, suggesting of basal EGFR phosphorylation only on the stiff, collagen-coated ECM-specific effects as well (Fig. S3B). Finally, western blot substrates (Fig. 2A,B). Increased EGFR phosphorylation on stiff analysis revealed higher levels of phosphorylated ERK1/2 (pERK) substrates prior to stimulation was also detected by and Akt (pAkt) – downstream targets of EGFR – on stiff PDMS

Fig. 2. Increased matrix stiffness stimulates EGF signaling. (A) Western blot analysis of pEGFR (Y1068) and total EGFR levels in keratinocytes cultured on 2% or 20% PDMS with or without collagen coating for 24 h in EGF-free medium, followed by stimulation with 10 ng/ml EGF for 15 min. (B) Quantification of the band intensity ratio for pEGFR:EGFR from cells on 2% or 20% collagen-coated PDMS prior to EGF stimulation. Data represent mean±s.e.m. (n=3 experiments), *P<0.05. (C) Immunofluorescence images of pEGFR (Y1068) in cells cultured on 2% or 20% PDMS at 0, 15, 30 or 60 min after stimulation with 10 ng/ml EGF. Scale bars: 20 µm. (D) Western blot analysis of pEGFR (Y1068) on PDMS substrates with 2, 5, 10 or 20% crosslinker. (E) Western blot analysis of additional EGFR phosphorylation sites (Y1068, Y845 or Y1173) on 2% or 20% PDMS. (F,G) Western blot analysis of downstream targets: pERK and total ERK (F), pAKT and total

AKT (G). For panels D–G, keratinocytes were cultured on collagen-coated PDMS substrates for 24 h in KSFM supplemented with 0.1 ng/ml EGF. Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780 substrates (Fig. 2F,G). We conclude that increased matrix stiffness Mechanical regulation of EGFR phosphorylation depends on promotes activation of the EGF signaling pathway in keratinocytes. focal adhesion signaling and cytoskeletal tension We next examined how stiffness-induced changes in EGF signaling EGF signaling mediates the proliferative response to matrix depended on mechanical linkage with the ECM. Consistent with stiffness previous findings (Trappmann et al., 2011), there were no To determine the functional role of EGF signaling in the growth measurable differences in cell spreading or organization of the response of keratinocytes to increased matrix stiffness, we F-actin cytoskeleton between keratinocytes cultured on 2% or 20% performed colony-forming assays on 2% or 20% PDMS PDMS substrates (Fig. 4A,B). However, cells on the stiff PDMS substrates with a range of different EGF concentrations. In the surfaces displayed significantly more focal adhesions detected by absence of exogenous EGF, keratinocytes formed colonies only on paxillin immunofluorescence (Fig. 4C,D). A similar response was the stiff PDMS substrates (Fig. 3A). At low EGF concentrations observed for vinculin, whereas there were no differences in overall (0.1 and 1 ng/ml), there were no differences in the number of expression of β1 integrin (Fig. S4). Moreover, paxillin expression, colonies formed, but colony size was significantly greater on the as well as focal adhesion kinase (FAK) phosphorylation at Y397, stiff, 20% PDMS (Fig. 3B,C). Colony size increased with increasing increased in a dose-dependent manner with increasing substrate EGF concentrations on the soft 2% substrates and, at the highest stiffness (Fig. 4E,F). Together, these results indicate that over this dose of 10 ng/ml, the difference in colony size between soft and stiff range of elastic moduli, substrate stiffening increases focal adhesion substrates was not statistically significant (Fig. 3B,C). Clonal number and total FAK activation. growth was completely blocked by treatment with the EGF inhibitor To investigate the crosstalk between focal adhesion and EGFR AG1478 (Fig. 3A,C). We conclude that EGF signaling mediates the signaling, we first assessed the direct interaction (within 30–40 nm) effects of matrix stiffness on keratinocyte proliferation. Moreover, a of EGFR with focal adhesions (i.e. paxillin) by using a proximity minimal level of EGF signaling is required by keratinocytes to ligation assay. There was a significantly higher interaction signal initiate colony formation but, at higher concentrations, EGF between EGFR and paxillin on 20% PDMS compared to 2% primarily regulates colony size. PDMS, and this relationship was reversed when acto-myosin We tested whether autocrine growth factor signaling was contractility was inhibited with Blebbistatin (Figs 5A,B and 4S). responsible for the increased growth on stiff substrates. Expansion Increased levels of phosphorylated EGFR (pEGFR) (Y1086) on of cells on PDMS substrates in the presence of conditioned medium 20% PDMS also colocalized with paxillin (Fig. S3A). Moreover, from keratinocytes on 2% or 20% PDMS enhanced overall clonal the disruption of cytoskeletal tension reversed the effects of growth, but there were no observable differences between the effects substrate stiffness on EGFR phosphorylation, with higher levels of medium from cells on either substrate (Fig. S3C). Similarly, there on 2% PDMS compared to 20% PDMS when treated with were no measurable differences in the level of amphiregulin, the Blebbistatin (Fig. 5C), while latrunculin treatment blocked the primary EGF-family ligand produced by keratinocytes (Piepkorn effects of substrate stiffness on EGFR phosphorylation and reduced et al., 1994), released into the medium by cells on 2% or 20% overall receptor expression (Fig. 5C). Additionally, we used siRNA substrates (Fig. S3D). These findings suggest that matrix stiffness to partially knock down vinculin and FAK (Fig. S4). Reduction in regulates EGFR activation through an intrinsic signaling total FAK led to higher EGFR phosphorylation on 2% PDMS mechanism rather than altered production of EGF ligands. compared to 20% PDMS, while reduced vinculin levels caused total

Fig. 3. EGF signaling mediates the growth response to matrix stiffness. (A) Representative images of Crystal Violet-stained keratinocyte colonies after 10 day clonal growth assays on 2% or 20% PDMS. Cells were treated with 0, 0.1, 1 or 10 ng/ml EGF, or 0.1 ng/ml EGF plus 10 µM of the EGFR inhibitor AG1478. (B,C) Quantification of colony number (B) and size (C) following exposure to EGF or the EGFR inhibitor. Data were normalized to 20% PDMS 0 ng/ml EGF levels and represent the mean±s.e.m. (n=3 experiments), *P<0.05 compared to 2% in identical medium. Journal of Cell Science

4 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780

Fig. 4. Substrate stiffness regulates focal adhesion assembly and signaling. (A) Representative images of phalloidin-stained F-actin in keratinocytes cultured on 2% or 20% substrates for 24 h. Scale bars: 50 µm. (B) Quantification of average cell area for keratinocytes on 2% or 20% PDMS. (C,D) Representative images of paxillin- containing focal adhesions (C) and quantification of average number of focal adhesions per cell (D). Scale bars: 20 µm. Data in B and D represent mean ±s.e.m. (n=3 experiments), *P<0.05. (E,F) Western blot analysis of paxillin (E), pFAK (Y397) (F) and total FAK in keratinocytes cultured on 2%, 5%, 10%, or 20% PDMS substrates.

EGFR to be downregulated, with no differences in phosphorylation modulus of the stress-strain curves at 30% strain was calculated as between soft and stiff substrates (Fig. 5D). Together, these results previously described (Delaine-Smith et al., 2016). The average indicate that the balance between ECM stiffness and F-actin modulus of normal skin samples was ∼6.1±2.9 kPa (mean±s.d.), cytoskeletal tension regulates the recruitment of EGFR to focal and the moduli of the keloid samples were significantly greater adhesions and that activation depends on FAK. (10×–100×), ranging from 50–650 kPa (Fig. 6B,C). Compared to Functionally, cytoskeletal tension and FAK activity were important our previous AFM analysis, the lower absolute values of moduli for keratinocyte growth in colony formation assays. Treatment with measured by micro-indentation were most likely to be due to a larger Blebbistatin reversed the effects of substrate stiffness on colony size, test area, which included the softer dermal tissue than the basement consistent with the effects on EGFR phosphorylation, while FAK membrane alone (Kao et al., 2016). inhibition with FAK inhibitor 14 completely blocked all colony In conjunction with mechanical testing, we analyzed EGFR formation (Fig. 5E). These results demonstrate that focal adhesion phosphorylation within the epidermis of keloid by assembly and signaling, as well as tension within the F-actin immunofluorescence staining and compared the levels of pEGFR cytoskeleton, are required for stiffness-dependent changes in EGFR to the extra-lesional (uninvolved) epidermis adjacent to the scar. phosphorylation and clonal growth in keratinocytes. Increased levels of pEGFR (Y0168) within the keloid scar could be observed in two out of three frozen sections examined, and levels of EGFR phosphorylation correlates with tissue stiffening in pEGFR (Y845) were ∼50% higher within the scarred skin across all keloid scars three patient samples (Fig. 6D,E). Consistent with our in vitro studies, Finally, to explore the physiologic significance of mechanically- these findings demonstrate that stiffening of the underlying in regulated EGF signaling within the skin we compared the keloids scars corresponds with EGFR activation in the epidermis and biomechanics of normal skin with stiff keloid scar tissue. Keloids suggest that tissue mechanics also regulates EGF signaling in vivo. are severe, injury-induced scars that expand beyond the initial wound margins and are characterized by excessive ECM production DISCUSSION and hyperproliferation (Andrews et al., 2016). They are believed to In the present study we employed silicone-based biomaterials with be stiffer than normal skin (Huang et al., 2016), but the mechanical tunable mechanical properties to investigate the effects of matrix properties have not been formally established yet. We performed stiffening on epidermal growth and differentiation. Our findings micro-indentation testing of the dermis of normal adult skin and the provide clear evidence that elevated matrix stiffness beyond normal expanding margins of keloid scars (Fig. 6A,B), and the tangent physiologic levels stimulates keratinocyte proliferation but does not Journal of Cell Science

5 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780

Fig. 5. Focal adhesion assembly and cytoskeletal tension are required for stiffness-dependent changes in EGF signaling. (A,B) Representative fluorescence images (A) and quantification of proximity ligation (PLA) interaction signal between paxillin and EGFR on 2% and 20% PDMS treated with 0.1% DMSO or 50 µM Blebbistatin (B). Scale bars: 25 µm. (C) Western blot analysis of pEGFR (Y1068) in keratinocytes cultured on 2% or 20% PDMS for 24 h while treated with 50 µM Blebbistatin, 1 µM latrunculin or carrier (0.1% DMSO) as a control. (D) Western blot analysis of pEGFR on 2% or 20% PDMS 72 h after transfection with siRNA targeting PTK2 (FAK) or VCL, or with non-targeting control (NTC) siRNA. (E) Quantification of colony size (normalized to 20% DMSO level) following 10 day exposure to 50 μM Blebbistatin or 416 nM FAK inhibitor 14. All data represent mean±s.e.m. (n=3 experiments), *P<0.05 compared to 2% DMSO; +P<0.05 compared to 20% DMSO. affect the ability to self-renew or terminally differentiate. Thus, and acto-myosin contractility. Moreover, this response involves matrix stiffness specifically regulates keratinocyte proliferation direct interaction between EGFR and focal adhesions, suggesting independently of effects on stem cell fate. Recent studies have that focal adhesion signaling molecules, such as FAK, may regulate shown that cultured human keratinocytes switch between expanding EGFR activity. However, it is also possible that EGFR activation and balanced modes of growth, which depend on cell-cell contact regulates focal adhesion assembly, and potential bi-directional and EGF signaling (Roshan et al., 2016). Our findings support this signaling and feedback mechanisms will be an important area of model and identify matrix stiffness as a key upstream regulator. In future investigation. Our findings are consistent with previous addition, another recent study demonstrated that increased matrix studies in normal and cancerous mammary epithelia (Klein et al., stiffness promotes directional migration of HaCaT keratinocytes 2009; Paszek et al., 2005; Wang et al., 1998), as well as recent (Wickert et al., 2016). Together, these results and our own establish findings, which link stiffness-dependent EGFR activation to a mechanism for how keratinocytes sense and respond to changes in Src-family kinases in fibroblasts (Saxena et al., 2017). Thus, bulk material elasticity. bio-mechanical regulation of focal adhesion assembly may play a Mechanistically, we show that EGF signaling mediates the key role in modulating EGF signaling across diverse cell types. growth response of human keratinocytes to altered matrix stiffness, In addition to cell-matrix adhesions, cell-cell adhesions also and phosphorylation of EGFR depends on focal adhesion assembly contribute to the biomechanical regulation of EGFRs. In simple Journal of Cell Science

6 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780

Fig. 6. EGFR activation correlates with tissue stiffness in vivo. (A) Representative H&E staining for a keloid scar. The dotted circle indicates the approximate location within the dermis of the keloid edge where micro-indentation tests were performed. (B) Representative load-displacement curves for individual keloid and normal skin samples indented up to 0.3 strain. (C) Quantification of tangent modulus at 0.3 strain for keloid scars and normal skin from a non-keloid affected donor; n=5 (normal) and =6 (keloid). *P<0.05. (D) Immunofluorescence staining for 14 (K14) and pEGFR (Y1068) in frozen sections of matched keloid and extra-lesional skin. Scale bars: 100 µm. (E) Quantification of pEGFR fluorescence intensity for tissue sections stained for pEGFR Y1068 or pEGFR Y845. Data are expressed as mean fluorescence intensity within the basal layer of the epidermis of the keloid relative to the basal layer of the extra-lesional epidermis from the same patient (n=3 donors). epithelia, increased substrate stiffness inhibits adherens junction tissue stiffness in and may play a role in keloid scar assembly, which in turn enhances EGF sensitivity and proliferation pathogenesis. While genome-wide association studies have only (Kim and Asthagiri, 2011). Recent studies also suggest that, in the linked a handful of genes to keloid susceptibility (Nakashima epidermis, elevated acto-myosin tension within adherens junctions et al., 2010), the underlying causes of keloid scar formation remain in the granular layer negatively regulates EGFR activity (Rübsam almost completely unknown. It is interesting to note that keloids et al., 2017). While our studies here aimed to establish the direct often develop in areas of skin with high tension (Andrews et al., signaling between the ECM and EGFR by using sparse, low-Ca2+ 2016; Huang et al., 2016; Ogawa et al., 2012), which combined culture conditions, the potential crosstalk with cell-cell adhesion with our findings, further supports a role for biomechanics in scar mechanics cannot be completely excluded. It will be interesting in formation. Future studies examining the inter- and intra-keloid future work to explore how biomechanical cues from both cell-ECM heterogeneity in mechanics, as well as the crosstalk with growth and cell-cell adhesions are integrated and regulate keratinocyte factor signaling and epidermal cell mechanics, will be important function under more confluent, in vivo-like conditions. Direct and will hopefully shed new light on these disfiguring and often measurement of forces at cell-cell adhesions (Borghi et al., 2012) painful conditions. and localization of activated EGFR within different mechanical environments will be of particular interest. Likewise, the role of MATERIALS AND METHODS additional cell adhesion receptors, such as desmosomes (Broussard Substrate preparation et al., 2017), and other growth factor receptors (Conway et al., 2013) PDMS substrates (Sylgard 184, Dow Corning) were prepared by mixing in cellular mechanosensing will be important areas of future the PDMS base with crosslinker at ratios varying from 50:1 (2%) to 5:1 investigation. Finally, it will also be necessary to consider the (20%). PDMS mixtures were de-gassed under a vacuum, spread onto 13 mm dynamics of focal adhesion turnover and EGFR recycling, as EGFR diameter glass coverslips or 6-well plates, and cured overnight at 70°C. To functionalize the PDMS substrates with ECM, the surfaces were covered is recycled together with β1 integrins (Caswell et al., 2008). with a solution of 50 mg/ml sulfo-SANPAH (Thermo Scientific) in water Although many studies have investigated the effects of substrate and exposed to 365 nm UV light for 10 min. This process was repeated in vitro mechanics on cell function using controlled models, only a twice, followed by incubation with either 50 μg/ml rat type I collagen (BD few have linked in vivo biomechanics to normal or pathological Biosciences) or 50 μg/ml human plasma fibronectin. Samples were rinsed functions (Gilbert et al., 2010; Levental et al., 2009). Our results three times with PBS, and sterilized with UVB and 70% ethanol prior to cell indicate that EGFR phosphorylation correlates with increased seeding. All chemicals were from Sigma-Aldrich unless otherwise noted. Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780

Cell culture development kit (R&D Systems, Abingdon, UK). Briefly, ELISA Primary human keratinocytes were isolated from neonatal foreskin and MaxiSorp 96-well plates (Thermo Scientific) were coated overnight at maintained on a layer of J2 3T3 fibroblasts in FAD medium as previously room temperature with the capture diluted in a 1% BSA/PBS described (Rheinwald and Green, 1977). For studies on PDMS substrates, solution. Plates were washed three times with 0.05% Tween 20 in PBS and fibroblasts were removed using Versene (Invitrogen), and keratinocytes blocked with 1% BSA for 1 h. Following a second wash step, 100 µl of the (passage 2–6) were trypsinized and seeded onto PDMS substrates in conditioned medium samples (pre-diluted 1:10) were incubated at room keratinocyte serum-free medium (KSFM, Invitrogen) supplemented with temperature for 2 h. The plate was washed three times, incubated with a bovine pituitary extract, penicillin/streptomycin, and 0.1 ng/ml EGF. To biotinylated anti-human amphiregulin antibody (from the kit) for 2 h at induce terminal differentiation, keratinocytes were cultured in KSFM room temperature, and washed three more times. Detection was performed further supplemented with 1.8 mM CaCl2. by incubation with 100 µl horseradish peroxidase (HRP)-tagged streptavidin for 20 min, followed by washing and incubation with the Tissue samples substrate solution for 20 min. The reaction was stopped with 50 µl Stop Skin samples were obtained from keloid patients and healthy volunteers Solution and read at 450 nm using a Synergy HT plate reader. from the plastic surgery department at Barts Health NHS Trust. All tissue samples were from dark skinned (South Asian or Afro-Caribbean) adult Immunofluorescence and imaging donors (male and female, under the age of 50); body sites included the back, For immunofluorescence staining, keratinocytes on PDMS substrates were shoulder, chest and stomach. All subjects gave informed consent and the fixed with 4% PFA and permeabilized with 0.1% Triton X-100 for 5 min. study was conducted under local ethical committee approval (East London Samples were blocked with 10% FBS plus 0.25% gelatin in PBS for 1 h and Research Ethics Committee, study no 2011-000626-29). incubated overnight at 4°C with primary against pEGFR Y1068 (as above, 1:500), pEGFR Y1086 [cat. no. ab32086, Abcam (1:500)], Colony-formation assay paxillin (as above, 1:500), vinculin [hVIN-1; cat. no. V9131, Sigma-Aldrich Primary keratinocytes were seeded onto non-ECM coated PDMS surfaces (1:1000)], E-cadherin [HECD1; cat. no. ab1416, Abcam (1:100)] or total within a 6-well plate at a density of 1000/well. Cells were cultured for EGFR (as above, 1:100). Secondary staining of pEGFR antibodies was 10 days in KSFM supplemented with 0–10 ng/ml EGF, 10 µM AG1478, performed with anti-rabbit AlexaFluor-488 [cat. no A11008, Thermo 50 µM Blebbistatin, or 416 nM FAK inhibitor 14 (Tocris Bioscience, Scientific (1:1000)], and anti-mouse AlexaFluor-568 or-488 [cat. nos Bristol, UK). Cells were fixed with 4% paraformaldehyde (PFA), stained for A10037 and A11001, Thermo Scientific (1:1000)]. F-actin was labeled with 30 min with 0.06% Crystal Violet, and rinsed copiously with water. phalloidin-AlexaFluor-568 or -488 (1:500, Sigma-Aldrich) included in the Scanned images of the stained wells were analyzed with ImageJ to quantify secondary solution. For the proximity ligation assay, fixed samples were colony size and number. co-stained with antibodies against paxillin and EGFR, and detected using the Duolink Mouse-Rabbit Red kit (Sigma-Aldrich) according to EdU labeling the manufacturer’s instructions. Samples were mounted on glass microscope To measure DNA synthesis, keratinocytes were cultured on PDMS slides with Mowiol and imaged using a Zeiss 710 confocal microscope (Carl substrates for 7 days in KSFM then incubated with 10 µM 5-ethynyl-2′- Zeiss) and 20× or 63× objectives. deoxyuridine (EdU) for 1 h at 37°C and rinsed twice with Edu-free KSFM. Normal skin and keloid tissue samples were either snap frozen in optimal Cells were fixed with 4% PFA, and EdU incorporated into the DNA was cutting temperature (OCT) compound (BD Biosciences) or fixed with 4% tagged with AlexaFluor-568 by using the ‘Click-It’ kit (Thermo Scientific) paraformaldehyde and embedded in paraffin. Freshly cut frozen sections according to the manufacturer’s instructions. Samples were co-stained with were fixed for 10 min in 4% paraformaldehyde, blocked with 10% FBS DAPI and imaged using a Leica DM5000B microscope. and 0.25% gelatin, and stained with anti-pEGFR Y1068 (as above, 1:300). Paraffin sections were de-waxed, and heat-mediated antigen retrieval was Western blot analysis performed with 10 mM sodium citrate. Sections were blocked as before and Cells were washed in PBS and incubated in radioimmunoprecipitation assay stained with anti-pEGFR Y845 (1:100, Cell Signaling, 6963S). Slides were (RIPA) buffer plus and phosphatase inhibitors for 10 min on ice. imaged with a Leica DM5000B microscope, and mean fluorescence Cells were scraped off the dish, briefly sonicated, and centrifuged (300 g for intensity of the basal layer was quantified with ImageJ. 5 min) to remove insoluble material. Protein concentration was determined by the BCA assay (Thermo Scientific). Lysates were combined with loading siRNA knockdown buffer (Thermo Scientific) and 1% β2-mercaptoethanol, and equal amounts Keratinocytes were seeded onto collagen-coated PDMS surfaces in a 6-well of total protein were resolved on a 4% or 10% polyacrylamide gel (Bio-Rad) plate and cultured overnight. Cells were transfected with 4 pmole siRNA and transferred onto nitrocellulose membranes (GE Lifesciences). and 4 µl of Jet Prime reagent (Polyplus Transfection) per well according to Membranes were blocked for 1 h in either 5% non-fat dry milk or 3% the manufacturer’s instructions. Cells were cultured for 72 h and harvested BSA, before being incubated either 1 h at room temperature or overnight at for western blot analysis. Silencer Select (Thermo Fisher) validated 4°C with primary antibodies against Involucrin [SY7, CRUK (1:1000)], small interfering RNAs (siRNAs) were used for PTK2 (s11486) and VCL pEGFR Y1068 [cat. no. 3777, Cell Signaling Technology, Tyr1068 (D7A5) (s14764) knockdown. Rb XP (1:1000)], EGFR [cat. no. 4267, Cell Signaling Technology (D38B1) Rb XP (1:1000)], pERK1/2 [cat. no. 9106S, Cell Signaling Technology Annexin V analysis (1:1000)], ERK1/2 [cat. no. 4695S, Cell Signaling Technology (1:1000)], and viability were analyzed by flow cytometry. Keratinocytes pAkt [cat. no. 9271, Cell Signaling Technology Ser473 Rb (1:1000)], Akt were cultured on PDMS substrates for 7 days, trypsinized, resuspended in [cat. no. 9272, Cell Signaling Technology Rb (1:1000)], Paxillin [cat. no. 200 µl Annexin V buffer (50 mM HEPES, 700 mM NaCl, 12.5 CaCl2) plus 610569, BD Biosciences (1:1000)], pFAK [cat. no. 611722, BD 5 µl Annexin V-FITC (Invitrogen) and DAPI, and incubated at room Biosciences (1:500)], FAK [cat. no. 0537, Millipore (1:500)] or GAPDH temperature for 15 min. Annexin V-positive (apoptotic) and DAPI-positive [cat. no. ab9485, Abcam Rb (1:2000)]. Secondary detection was performed (dead) cells were analyzed by flow cytometry using the Becton Dickinson with HRP-conjugated anti-rabbit or anti-mouse antibodies (1:5000, Dako). LSRII. As a positive control for apoptosis, cells were treated with 10 mJ/cm2 were visualized using the enhanced chemiluminescence detection UVB light 20 h prior to analysis. system (Millipore, Watford, UK).

ELISA Material characterization and mechanical testing The -linked immunosorbent assay (ELISA) for amphiregulin was For the mechanical testing of the PDMS, samples were cast into Petri dishes performed on conditioned medium from cells cultured on PDMS substrates and, after curing were cut to an approximate size of 5 mm wide×25 mm for 24 h according to the manufacturer’s protocol for the DuoSet ELISA long×1.5 mm thick. Tensile testing was conducted using an Instron Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780 universal testing system. The PDMS samples were held in place using References pneumatic grips and tested to failure at a strain rate of 20 mm/min. The Adams, J. C. and Watt, F. M. (1989). Fibronectin inhibits the terminal differentiation elastic modulus was calculated from the slope of the stress versus strain of human keratinocytes. Nature 340, 307-309. Andrews, J. P., Marttala, J., Macarak, E., Rosenbloom, J. and Uitto, J. (2016). curve. Six samples per setting (2% and 20%) were tested. Keloids: the paradigm of skin fibrosis-Pathomechanisms and treatment. Matrix For scanning electron microscopy (SEM) analysis of surface topography, Biol. 51, 37-46. cover slips were coated with PDMS and gold-coated using an Agar auto Assefa, Z., Garmyn, M., Bouillon, R., Merlevede, W., Vandenheede, J. R. and sputter coater. SEM imaging was conducted using an FEI Inspect-F with an Agostinis, P. (1997). Differential stimulation of ERK and JNK activities by accelerating voltage of 5 kV and a working distance of 10 mm. The B irradiation and epidermal growth factor in human keratinocytes. roughness measurements were performed using AFM (nTegra, NT-MDT). J. Invest. Dermatol. 108, 886-891. μ Blanpain, C. and Fuchs, E. (2009). Epidermal homeostasis: a balancing act of stem The samples were imaged over a 10×10 m area in semi-contact mode with cells in the skin. Nat. Rev. Mol. Cell Biol. 10, 207-217. a silicon nitride cantilever (MLCT, Bruker, spring constant k=0.6 N/m). The Borghi, N., Sorokina, M., Shcherbakova, O. G., Weis, W. I., Pruitt, B. L., Nelson, sample height at each point within the image and average roughness were W. J. and Dunn, A. R. (2012). E-cadherin is under constitutive actomyosin- measured using the AFM software (Nova, NT-MDT). generated tension that is increased at cell–cell contacts upon externally applied The tissue moduli of keloid and normal skin samples were measured by stretch. Proc. Natl. Acad. Sci. USA 109, 12568-12573. micro-indentation. Frozen specimens were fully thawed at room temperature Broussard, J. A., Yang, R., Huang, C., Nathamgari, S. S. P., Beese, A. M., Godsel, L. M., Hegazy, M. H., Lee, S., Zhou, F., Sniadecki, N. J. et al. (2017). in PBS for 1 h before testing. Mechanical indentation was performed using The desmoplakin-intermediate filament linkage regulates cell mechanics. Mol. an Instron ElectroPuls E1000 (Instron) equipped with a 10 N load cell Biol. Cell 28, 3156-3164. (resolution=0.1 mN). Specimens were indented using a stainless steel plane- Bruckner-Tuderman, L., Mitsuhashi, Y., Schnyder, U. W. and Bruckner, P. ended cylindrical punch with a diameter (Øi) of 2 or 1 mm. Specimen (1989). Anchoring fibrils and type VII collagen are absent from skin in severe recessive dystrophic epidermolysis bullosa. J. Invest. Dermatol. 93, 3-9. thickness (Ts) was measured as the distance between the base of the test dish Caswell, P. T., Chan, M., Lindsay, A. J., McCaffrey, M. W., Boettiger, D. and and top of the sample, each detected by applying a pre-load of 0.3 mN. Ø Norman, J. C. (2008). Rab-coupling protein coordinates recycling of alpha5beta1 Specimen diameter ( s) was measured using electronic callipers. integrin and EGFR1 to promote cell migration in 3D microenvironments. J. Cell Indentation was performed at room temperature with specimens fully Biol. 183, 143-155. submerged in PBS throughout testing. Tests were performed using a ramped Chowdhury, F., Li, Y., Poh, Y.-C., Yokohama-Tamaki, T., Wang, N. and Tanaka, displacement-control regime whereby each specimen was displaced to 30% T. S. (2010). Soft substrates promote homogeneous self-renewal of embryonic of their measured thickness at a rate of 1% s−1. The resulting load detected stem cells via downregulating cell-matrix tractions. PLoS ONE 5, e15655. Connelly, J. T., Gautrot, J. E., Trappmann, B., Tan, D. W.-M., Donati, G., Huck, from the sample was recorded at 10 Hz. To minimise errors in calculations W. T. S. and Watt, F. M. (2010). Actin and serum response factor transduce of tissue moduli, specimen to indenter ratios were kept to Øs:Øi ≥4:1 and physical cues from the microenvironment to regulate epidermal stem cell fate Ts:Øi ≤2:1. decisions. Nat. Cell Biol. 12, 711-718. Tissue moduli were calculated from the tangents of the final linear regions Conway, D. E., Breckenridge, M. T., Hinde, E., Gratton, E., Chen, C. S. and of the load-displacement experimental data with the aid of a corrected Schwartz, M. A. (2013). Fluid shear stress on endothelial cells modulates mathematical model (Delaine-Smith et al., 2016). Briefly, the tissue mechanical tension across VE-cadherin and PECAM-1. Curr. Biol. 23, E S 1024-1030. modulus ( ) is related to the indentation contact stiffness ( ) and the radius Delaine-Smith, R. M., Burney, S., Balkwill, F. R. and Knight, M. M. (2016). of the flat punch indenter (a) by the following relationship: Experimental validation of a flat punch indentation methodology calibrated against unconfined compression tests for determination of soft tissue biomechanics. E ¼½ðS= akÞ=G ð n2Þ: ð Þ J. Mech. Behav. Biomed. Mater. 60, 401-415. 2 k 1 1 Gat, U., DasGupta, R., Degenstein, L. and Fuchs, E. (1998). De Novo follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in The geometric correction factor к accounts for large-deformation, non- skin. Cell 95, 605-614. linear behavior and values for strains >15% can be determined from linear Gilbert, P. M., Havenstrite, K. L., Magnusson, K. E. G., Sacco, A., Leonardi, interpolation (Zhang et al., 1997). The second geometrical correction factor, N. A., Kraft, P., Nguyen, N. K., Thrun, S., Lutolf, M. P. and Blau, H. M. (2010). G ’ ν Substrate elasticity regulates stem cell self-renewal in culture. к, is applied from Delaine-Smith et al. (2016). Poisson s ratio ( ), was Science 329, 1078-1081. assumed to be 0.499 for all specimens. Green, K. J. and Simpson, C. L. (2007). Desmosomes: new perspectives on a classic. J. Invest. Dermatol. 127, 2499-2515. Statistical analysis Herrick, S. E., Sloan, P., McGurk, M., Freak, L., McCollum, C. N. and Ferguson, All data were analyzed by ANOVA and Tukey’s test for post-hoc analysis M. W. (1992). Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers. Am. J. Pathol. 141, with sample size of independent experiments or patients indicated in the 1085-1095. figure captions. Huang, C., Liu, L., You, Z., Wang, B., Du, Y. and Ogawa, R. (2016). Keloid progression: a stiffness gap hypothesis. Int. Wound J. 14, 764-771. Acknowledgements Jones, P. H. and Watt, F. M. (1993). Separation of human epidermal stem cells from We thank Dr Gary Warnes for assistance with annexin V analysis and Oscar Pundel transit amplifying cells on the basis of differences in integrin function and for maintenance of primary keratinocyte cultures. expression. Cell 73, 713-724. Jost, M., Kari, C. and Rodeck, U. (2000). The EGF receptor-an essential regulator of multiple epidermal functions. Eur. J. Dermatol. 10, 505-510. Competing interests Kao, A. P., Connelly, J. T. and Barber, A. H. (2016). 3D nanomechanical Fiona Kenny has carried out paid consultancy work for Metaphase Ltd. evaluations of dermal structures in skin. J. Mech. Behav. Biomed. Mater. 57, 14-23. Author contributions Kim, J.-H. and Asthagiri, A. R. (2011). Matrix stiffening sensitizes epithelial cells to Conceptualization: J.T.C.; Formal analysis: F.N.K., R.D.-S., A.P.K.; Investigation: EGF and enables the loss of contact inhibition of proliferation. J. Cell. Sci. 124, F.N.K., Z.D., R.D.-S., A.P.K., A.C.L., J.T.C.; Resources: Z.D., M.P.P.; Writing - 1280-1287. original draft: F.N.K., J.T.C.; Writing - review & editing: J.T.C.; Supervision: M.M.K., Klein, E. A., Yin, L., Kothapalli, D., Castagnino, P., Byfield, F. J., Xu, T., Levental, M.P.P., J.T.C.; Project administration: M.M.K.; Funding acquisition: J.T.C. I., Hawthorne, E., Janmey, P. A. and Assoian, R. K. (2009). Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening. Curr. Biol. 19, Funding 1511-1518. Lee-Thedieck, C., Rauch, N., Fiammengo, R., Klein, G. and Spatz, J. P. (2012). This work was funded by the Barts Charity (Large Grant 442/1032), the British Skin Impact of substrate elasticity on human hematopoietic stem and progenitor cell Foundation (PhD studentship grant no: 4052s for F.N.K.), and the European adhesion and motility. J. Cell Sci. 125, 3765-3775. Research Council (CANBUILD project 322566 for R.D.-S.). Levental, K. R., Yu, H., Kass, L., Lakins, J. N., Egeblad, M., Erler, J. T., Fong, S. F. T., Csiszar, K., Giaccia, A., Weninger, W. et al. (2009). Matrix crosslinking Supplementary information forces tumor progression by enhancing integrin signaling. Cell 139, 891-906. Supplementary information available online at Martins, V. L., Vyas, J. J., Chen, M., Purdie, K., Mein, C. A., South, A. P., Storey, http://jcs.biologists.org/lookup/doi/10.1242/jcs.215780.supplemental A., McGrath, J. A. and O’Toole, E. A. (2009). Increased invasive behaviour in Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs215780. doi:10.1242/jcs.215780

cutaneous squamous cell carcinoma with loss of basement-membrane type VII Saxena, M., Liu, S., Yang, B., Hajal, C., Changede, R., Hu, J., Wolfenson, H., collagen. J. Cell. Sci. 122, 1788-1799. Hone, J. and Sheetz, M. P. (2017). EGFR and HER2 activate rigidity sensing only Nakashima, M., Chung, S., Takahashi, A., Kamatani, N., Kawaguchi, T., on rigid matrices. Nat. Mater. 16, 775-781. Tsunoda, T., Hosono, N., Kubo, M., Nakamura, Y. and Zembutsu, H. (2010). Stojadinovic, O., Brem, H., Vouthounis, C., Lee, B., Fallon, J., Stallcup, M., A genome-wide association study identifies four susceptibility loci for keloid in the Merchant, A., Galiano, R. D. and Tomic-Canic, M. (2005). Molecular Japanese population. Nat. Genet. 42, 768-771. pathogenesis of chronic wounds: the role of beta-catenin and c-myc in the Niessen, C. M. (2007). Tight junctions/adherens junctions: basic structure and inhibition of epithelialization and . Am. J. Pathol. 167, 59-69. function. J. Invest. Dermatol. 127, 2525-2532. Trappmann, B., Gautrot, J. E., Connelly, J. T., Strange, D. G., Li, Y., Oyen, M. L., Ogawa, R., Okai, K., Tokumura, F., Mori, K., Ohmori, Y., Huang, C., Hyakusoku, Cohen Stuart, M. A., Boehm, H., Li, B., Vogel, V. et al. (2011). Extracellular- H. and Akaishi, S. (2012). The relationship between skin stretching/contraction matrix tethering regulates stem-cell fate. Nat. Mater. 11, 642-649. and pathologic scarring: the important role of mechanical forces in keloid Uribe, P. and Gonzalez, S. (2011). Epidermal growth factor receptor (EGFR) and generation. Wound Repair. Regen. 20, 149-157. squamous cell carcinoma of the skin: molecular bases for EGFR-targeted Paszek, M. J., Zahir, N., Johnson, K. R., Lakins, J. N., Rozenberg, G. I., Gefen, A., therapy. Pathol. Res. Pract. 207, 337-342. Reinhart-King, C. A., Margulies, S. S., Dembo, M., Boettiger, D. et al. (2005). Wan, Y. S., Wang, Z. Q., Shao, Y., Voorhees, J. J. and Fisher, G. J. (2001). Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241-254. Ultraviolet irradiation activates PI 3-kinase/AKT survival pathway via EGF Piepkorn, M. (1996). Overexpression of amphiregulin, a major autocrine growth receptors in human skin in vivo. Int. J. Oncol. 18, 461-466. factor for cultured human keratinocytes, in hyperproliferative skin diseases. Wang, F., Weaver, V. M., Petersen, O. W., Larabell, C. A., Dedhar, S., Briand, P., Am. J. Dermatopathol. 18, 165-171. Lupu, R. and Bissell, M. J. (1998). Reciprocal interactions between beta1- Piepkorn, M., Lo, C. and Plowman, G. (1994). Amphiregulin-dependent integrin and epidermal growth factor receptor in three-dimensional basement proliferation of cultured human keratinocytes: autocrine growth, the effects of exogenous recombinant cytokine, and apparent requirement for heparin-like membrane breast cultures: a different perspective in epithelial biology. Proc. Natl. glycosaminoglycans. J. Cell. Physiol. 159, 114-120. Acad. Sci. USA 95, 14821-14826. Reiss, M. and Sartorelli, A. C. (1987). Regulation of growth and differentiation of Wickert, L. E., Pomerenke, S., Mitchell, I., Masters, K. S. and Kreeger, P. K. human keratinocytes by type beta transforming growth factor and epidermal (2016). Hierarchy of cellular decisions in collective behavior: implications for growth factor. Cancer Res. 47, 6705-6709. wound healing. Sci. Rep. 6, 20139. Rheinwald, J. G. and Green, H. (1977). Epidermal growth factor and the Wysocki, A. B., Staiano-Coico, L. and Grinnell, F. (1993). Wound fluid from multiplication of cultured human epidermal keratinocytes. Nature 265, 421-424. chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and Roshan, A., Murai, K., Fowler, J., Simons, B. D., Nikolaidou-Neokosmidou, V. MMP-9. J. Invest. Dermatol. 101, 64-68. and Jones, P. H. (2016). Human keratinocytes have two interconvertible modes Zhang, M., Zheng, Y. P. and Mak, A. F. T. (1997). Estimating the effective Young’s of proliferation. Nat. Cell Biol. 18, 145-156. modulus of soft tissues from indentation tests–nonlinear finite element analysis of Rübsam, M., Mertz, A. F., Kubo, A., Marg, S., Jüngst, C., Goranci-Buzhala, G., effects of friction and large deformation. Med. Eng. Phys. 19, 512-517. Schauss, A. C., Horsley, V., Dufresne, E. R., Moser, M. et al. (2017). E-cadherin Zhu, A. J. and Watt, F. M. (1999). beta-catenin signalling modulates proliferative integrates mechanotransduction and EGFR signaling to control junctional tissue potential of human epidermal keratinocytes independently of intercellular polarization and tight junction positioning. Nat. Commun. 8, 1250. adhesion. Development 126, 2285-2298. Journal of Cell Science

10