RESEARCH ARTICLE

Tissue stiffening promotes keratinocyte proliferation throughactivation of epidermal growth factor signalingFiona 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,*

ABSTRACTTissue biomechanics regulate a wide range of cellular functions, butthe influences on epidermal homeostasis and repair remain unclear.Here, we examined the role of extracellular matrix stiffness on humankeratinocyte behavior using elastomeric substrates with definedmechanical properties. Increased matrix stiffness beyond normalphysiologic levels promoted keratinocyte proliferation but did not alterthe ability to self-renew or terminally differentiate. Activation ofepidermal growth factor (EGF) signaling mediated the proliferativeresponse to matrix stiffness and depended on focal adhesionassembly and cytoskeletal tension. Comparison of normal skin withkeloid scar tissue further revealed an upregulation of EGF signalingwithin the epidermis of stiffened scar tissue. We conclude that matrixstiffness regulates keratinocyte proliferation independently of changesin cell fate and is mediated by EGF signaling. These findings providemechanistic insights into how keratinocytes sense and respond to theirmechanical environment, and suggest that matrix biomechanics mayplay a role in the pathogenesis keloid scar formation.

KEY WORDS: Mechanotransduction, Keratinocyte, Epidermis, EGF,Keloid, Proliferation

INTRODUCTIONIn the epidermis of the skin, the balance between keratinocyteproliferation in the basal layer and terminal differentiation andshedding in the upper layers maintains normal tissue homeostasis(Blanpain and Fuchs, 2009). These processes depend on a variety ofextracellular cues and signals, such as soluble growth factors (Reissand Sartorelli, 1987; Rheinwald and Green, 1977; Zhu and Watt,1999), cell-cell adhesion (Green and Simpson, 2007; Niessen, 2007),and cell-extracellular matrix (ECM) interactions (Adams and Watt,1989; Jones andWatt, 1993). Dysregulation of key extrinsic signalingpathways can lead to an imbalance in growth and differentiation andoften contributes to the pathogenesis of skin diseases includingchronic wounds (Herrick et al., 1992; Stojadinovic et al., 2005;Wysocki et al., 1993), blistering (Bruckner-Tuderman et al., 1989),and cancer progression (Gat et al., 1998; Martins et al., 2009; Reissand Sartorelli, 1987; Uribe and Gonzalez, 2011).Epidermal growth factor (EGF) signaling is one of the major

regulatory axes controlling keratinocyte proliferation and survival.

The EGF receptor (EGFR) is a receptor tyrosine kinase that is highlyexpressed in the basal layer of the epidermis and, upon binding ofEGF ligands, such as EGF, amphiregulin and transforming growthfactor α (TGF-α), the receptor dimerizes and becomes activated byautophosphorylation at multiple tyrosine (Y) residues (Jost et al.,2000). Under homeostatic conditions, EGF signaling promotesgrowth and survival of basal keratinocytes through downstreamactivation of mitogen activated protein kinase (MAPK) andphosphotidylinositide 3-kinase (PI3K) signaling pathways (Assefaet al., 1997;Wan et al., 2001). However, overexpression of EGFR orits ligands is associated with a variety of hyperproliferativeconditions, such as psoriasis (Piepkorn, 1996) and cancer (Reissand Sartorelli, 1987; Uribe and Gonzalez, 2011).

While the roles of many biochemical factors in the regulation ofkeratinocyte function have been described in detail, little is knownabout the contribution of mechanical or biophysical cues. In ourprevious studies, we used micro-patterned substrates and establishedthat simple changes in keratinocyte shape and adhesion are potentregulators of terminal differentiation (Connelly et al., 2010).Similarly, reduced tethering of ECM molecules to cell culturesupports can induce terminal differentiation (Trappmann et al., 2011).While bulk material stiffness appears to have little effect onkeratinocyte differentiation, the impact on additional cell functionsor fate over longer time scales has yet to be determined. As tissuestiffness regulates the proliferation and self-renewal of multiple celltypes, including mammary epithelia (Klein et al., 2009; Paszek et al.,2005), muscle-derived stem cells (Gilbert et al., 2010), hematopoieticstem cells (Lee-Thedieck et al., 2012) and mesenchymal stem cells(Chowdhury et al., 2010), it may also be an important mediator ofepidermal keratinocyte growth.

In the present study we investigated the effects of altered matrixstiffness on keratinocyte behavior using model silicone substrates.We show that increased matrix stiffness promotes epidermalproliferation independently of changes in cell fate, and that EGFsignaling mediates this response. We also demonstrate that EGFsignaling is elevated within keloid scar tissue, which is ∼30-foldstiffer than normal skin. These findings provide significant insightsinto the mechanisms of mechanosensing within the epidermis, andtheir impact on tissue homeostasis and scar formation.

RESULTSSubstrate stiffness regulates keratinocyte proliferationindependently of cell fateTo investigate the influence of matrix stiffness on long-termkeratinocyte growth and differentiation, we generated cell culturesubstrates with defined elastic moduli using polydimethylsiloxane(PDMS). PDMS substrates were crosslinked with 2% or 20%(w/w) curing agent to produce non-porous substrates with elasticmoduli of 180 kPa or 2 MPa, respectively (Fig. S1). Our previousatomic force microscopy (AFM) analysis of normal skin measuredReceived 22 January 2018; Accepted 11 April 2018

1Centre for Cell Biology and Cutaneous Research, Barts and the London School ofMedicine and Dentistry, Queen Mary University of London, London E1 2AT, UK.2School of Engineering and Materials Science, Queen Mary University of London,London E1 4NS, UK. 3Institute of Bioengineering, QueenMary University of London,London E1 4NS, UK.

*Author for correspondence ( j.connelly@qmul.ac.uk)

J.T.C., 0000-0002-5955-8848

1

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

Journal

ofCe

llScience

the elastic modulus of the basement membrane to be ∼140 kPa(Kao et al., 2016). Thus, the PDMS substrates with 2% curingagent were most similar to normal skin, while the substratescrosslinked with 20% curing agent represented a ten-fold increasein matrix stiffness.Primary human keratinocytes were seeded onto 2% or 20%

PDMS substrates at clonal density and cultured for 10 days in low-Ca2+, serum-free medium with 0.1 ng/ml EGF. Cells formed asimilar number of colonies on both substrates but the colonies on thestiff 20% substrates were significantly larger (Fig. 1A,C). Trackingof individual colonies over the first seven days revealed a morerapid, exponential increase in the number of cells per clone on the20% substrates compared to the 2% substrates (Fig. 1D), andkeratinocytes on the stiff substrates had a higher proliferative rate atday 7 (Fig. 1E,F). There were no detectable differences in initialadhesion or viability of keratinocyte cultured on the soft and stiffsubstrates (Fig. S2).

To assess whether substrate stiffness influenced epidermal cellfate, keratinocytes were first expanded clonally on 2% or 20%PDMS for 10 days, then dissociated and expanded a second time on2% or 20% PDMS. Keratinocytes formed similar numbers ofcolonies under all conditions (Fig. 1G,H), indicating that previousexposure to a soft or stiff environment did not affect the proportionof colony-initiating cells within the culture, a common read-out ofepidermal stem cell function in vitro (Jones and Watt, 1993). Inaddition, expression of the terminal differentiation markerinvolucrin, was similar for cells cultured on 2% or 20% PDMSfor 5 days followed by stimulation with Ca2+ (1.8 mM CaCl2) for2 days to induce terminal differentiation (Fig. 1I). Likewise, therewere no striking differences in Ca2+-induced assembly of adherensjunctions over this range of substrate moduli (Fig. S4). Takentogether, these findings indicate that increased substrate stiffnessspecifically stimulates keratinocyte proliferation but does not affectadhesion, 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 for10 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) fromscanned 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 colonyon 2% and 20% PDMS based on bright-field images (10×) at defined locations, tracked from day 3–7. (E,F) Representative images and quantification ofEdU-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=4experiments), *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 representmean±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 for5 days in KSFM followed by stimulation with 1.8 mM CaCl2 for 2 days.

2

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

Journal

ofCe

llScience

matrix stiffness regulates keratinocyte growth independently ofchanges in cell fate.

Increased substrate stiffness activates EGF signalingTo gain insight into the mechanism by which matrix stiffnessregulates keratinocyte proliferation we first examined the effects onEGF signaling, a key regulator of proliferation and survival (Jostet al., 2000). Cells were cultured on 2%or 20%PDMS substrates withor without collagen coating for 24 h in EGF-free medium, thenstimulated with 10 ng/ml EGF for 15 min. High levels of EGFRphosphorylation at Y1068 were observed on all substrates followingEGF stimulation (Fig. 2A), but there was a significantly higher levelof basal EGFR phosphorylation only on the stiff, collagen-coatedsubstrates (Fig. 2A,B). Increased EGFR phosphorylation on stiffsubstrates prior to stimulation was also detected by

immunofluorescence staining, while receptor internalizationfollowing treatment with EGF appeared to be unaffected bysubstrate stiffness (Fig. 2C). A similar increase in the basal level ofphosphorylated Y845, Y1086 and Y1173 was also observed on thestiff substrates (Fig. 2D, Fig. S3A).

The effects of matrix stiffness on EGF signaling occurred in adose-dependent manner. EGFR phosphorylation progressivelyincreased with increasing substrate stiffness for PDMS crosslinkedwith 2%, 5%, 10% or 20% curing agent (Fig. 2D). There was alsoelevated EGFR phosphorylation in keratinocytes cultured on stiffsubstrates coated with fibronectin; however, the response onfibronectin was associated with increased total EGFR, suggestingECM-specific effects as well (Fig. S3B). Finally, western blotanalysis revealed higher levels of phosphorylated ERK1/2 (pERK)and Akt (pAkt) – downstream targets of EGFR – on stiff PDMS

Fig. 2. Increasedmatrix stiffness stimulatesEGFsignaling. (A)Western blot analysis of pEGFR (Y1068) and total EGFR levels in keratinocytes cultured on 2%or20% 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 bandintensity 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 EGFRphosphorylation 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 totalAKT (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.

3

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

Journal

ofCe

llScience

substrates (Fig. 2F,G). We conclude that increased matrix stiffnesspromotes activation of the EGF signaling pathway in keratinocytes.

EGF signaling mediates the proliferative response to matrixstiffnessTo determine the functional role of EGF signaling in the growthresponse of keratinocytes to increased matrix stiffness, weperformed colony-forming assays on 2% or 20% PDMSsubstrates with a range of different EGF concentrations. In theabsence of exogenous EGF, keratinocytes formed colonies only onthe stiff PDMS substrates (Fig. 3A). At low EGF concentrations(0.1 and 1 ng/ml), there were no differences in the number ofcolonies formed, but colony size was significantly greater on thestiff, 20% PDMS (Fig. 3B,C). Colony size increased with increasingEGF concentrations on the soft 2% substrates and, at the highestdose of 10 ng/ml, the difference in colony size between soft and stiffsubstrates was not statistically significant (Fig. 3B,C). Clonalgrowth was completely blocked by treatment with the EGF inhibitorAG1478 (Fig. 3A,C). We conclude that EGF signaling mediates theeffects of matrix stiffness on keratinocyte proliferation. Moreover, aminimal level of EGF signaling is required by keratinocytes toinitiate colony formation but, at higher concentrations, EGFprimarily regulates colony size.We tested whether autocrine growth factor signaling was

responsible for the increased growth on stiff substrates. Expansionof cells on PDMS substrates in the presence of conditioned mediumfrom keratinocytes on 2% or 20% PDMS enhanced overall clonalgrowth, but therewere no observable differences between the effectsof medium from cells on either substrate (Fig. S3C). Similarly, therewere no measurable differences in the level of amphiregulin, theprimary EGF-family ligand produced by keratinocytes (Piepkornet al., 1994), released into the medium by cells on 2% or 20%substrates (Fig. S3D). These findings suggest that matrix stiffnessregulates EGFR activation through an intrinsic signalingmechanism rather than altered production of EGF ligands.

Mechanical regulation of EGFR phosphorylation depends onfocal adhesion signaling and cytoskeletal tensionWe next examined how stiffness-induced changes in EGF signalingdepended on mechanical linkage with the ECM. Consistent withprevious findings (Trappmann et al., 2011), there were nomeasurable differences in cell spreading or organization of theF-actin cytoskeleton between keratinocytes cultured on 2% or 20%PDMS substrates (Fig. 4A,B). However, cells on the stiff PDMSsurfaces displayed significantly more focal adhesions detected bypaxillin immunofluorescence (Fig. 4C,D). A similar response wasobserved for vinculin, whereas there were no differences in overallexpression of β1 integrin (Fig. S4). Moreover, paxillin expression,as well as focal adhesion kinase (FAK) phosphorylation at Y397,increased in a dose-dependent manner with increasing substratestiffness (Fig. 4E,F). Together, these results indicate that over thisrange of elastic moduli, substrate stiffening increases focal adhesionnumber and total FAK activation.

To investigate the crosstalk between focal adhesion and EGFRsignaling, we first assessed the direct interaction (within 30–40 nm)of EGFR with focal adhesions (i.e. paxillin) by using a proximityligation assay. There was a significantly higher interaction signalbetween EGFR and paxillin on 20% PDMS compared to 2%PDMS, and this relationship was reversed when acto-myosincontractility was inhibited with Blebbistatin (Figs 5A,B and 4S).Increased levels of phosphorylated EGFR (pEGFR) (Y1086) on20% PDMS also colocalized with paxillin (Fig. S3A). Moreover,the disruption of cytoskeletal tension reversed the effects ofsubstrate stiffness on EGFR phosphorylation, with higher levelson 2% PDMS compared to 20% PDMS when treated withBlebbistatin (Fig. 5C), while latrunculin treatment blocked theeffects of substrate stiffness on EGFR phosphorylation and reducedoverall receptor expression (Fig. 5C). Additionally, we used siRNAto partially knock down vinculin and FAK (Fig. S4). Reduction intotal FAK led to higher EGFR phosphorylation on 2% PDMScompared to 20% PDMS, while reduced vinculin levels caused total

Fig. 3. EGF signaling mediates thegrowth response to matrix stiffness.(A) Representative images of CrystalViolet-stained keratinocyte coloniesafter 10 day clonal growth assays on2% or 20% PDMS. Cells were treatedwith 0, 0.1, 1 or 10 ng/ml EGF, or0.1 ng/ml EGF plus 10 µM of the EGFRinhibitor AG1478. (B,C) Quantificationof colony number (B) and size (C)following exposure to EGF or theEGFR inhibitor. Data were normalizedto 20% PDMS 0 ng/ml EGF levels andrepresent the mean±s.e.m. (n=3experiments), *P<0.05 compared to2% in identical medium.

4

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

Journal

ofCe

llScience

EGFR to be downregulated, with no differences in phosphorylationbetween soft and stiff substrates (Fig. 5D). Together, these resultsindicate that the balance between ECM stiffness and F-actincytoskeletal tension regulates the recruitment of EGFR to focaladhesions and that activation depends on FAK.Functionally, cytoskeletal tension and FAK activity were important

for keratinocyte growth in colony formation assays. Treatment withBlebbistatin reversed the effects of substrate stiffness on colony size,consistent with the effects on EGFR phosphorylation, while FAKinhibition with FAK inhibitor 14 completely blocked all colonyformation (Fig. 5E). These results demonstrate that focal adhesionassembly and signaling, as well as tension within the F-actincytoskeleton, are required for stiffness-dependent changes in EGFRphosphorylation and clonal growth in keratinocytes.

EGFR phosphorylation correlates with tissue stiffening inkeloid scarsFinally, to explore the physiologic significance of mechanically-regulated EGF signaling within the skin we compared thebiomechanics of normal skin with stiff keloid scar tissue. Keloidsare severe, injury-induced scars that expand beyond the initialwound margins and are characterized by excessive ECM productionand hyperproliferation (Andrews et al., 2016). They are believed tobe stiffer than normal skin (Huang et al., 2016), but the mechanicalproperties have not been formally established yet. We performedmicro-indentation testing of the dermis of normal adult skin and theexpanding margins of keloid scars (Fig. 6A,B), and the tangent

modulus of the stress-strain curves at 30% strain was calculated aspreviously described (Delaine-Smith et al., 2016). The averagemodulus of normal skin samples was ∼6.1±2.9 kPa (mean±s.d.),and the moduli of the keloid samples were significantly greater(10×–100×), ranging from 50–650 kPa (Fig. 6B,C). Compared toour previous AFM analysis, the lower absolute values of modulimeasured bymicro-indentation were most likely to be due to a largertest area, which included the softer dermal tissue than the basementmembrane alone (Kao et al., 2016).

In conjunction with mechanical testing, we analyzed EGFRphosphorylation within the epidermis of keloid scars byimmunofluorescence staining and compared the levels of pEGFRto the extra-lesional (uninvolved) epidermis adjacent to the scar.Increased levels of pEGFR (Y0168) within the keloid scar could beobserved in two out of three frozen sections examined, and levels ofpEGFR (Y845) were ∼50% higher within the scarred skin across allthree patient samples (Fig. 6D,E). Consistent with our in vitro studies,these findings demonstrate that stiffening of the underlying dermis inkeloids scars corresponds with EGFR activation in the epidermis andsuggest that tissue mechanics also regulates EGF signaling in vivo.

DISCUSSIONIn the present study we employed silicone-based biomaterials withtunable mechanical properties to investigate the effects of matrixstiffening on epidermal growth and differentiation. Our findingsprovide clear evidence that elevated matrix stiffness beyond normalphysiologic levels stimulates keratinocyte proliferation but does not

Fig. 4. Substrate stiffness regulatesfocal adhesion assembly andsignaling. (A) Representative images ofphalloidin-stained F-actin in keratinocytescultured on 2% or 20% substrates for24 h. Scale bars: 50 µm.(B) Quantification of average cell area forkeratinocytes on 2% or 20% PDMS.(C,D) Representative images of paxillin-containing focal adhesions (C) andquantification of average number of focaladhesions 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 inkeratinocytes cultured on 2%, 5%, 10%,or 20% PDMS substrates.

5

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

Journal

ofCe

llScience

affect the ability to self-renew or terminally differentiate. Thus,matrix stiffness specifically regulates keratinocyte proliferationindependently of effects on stem cell fate. Recent studies haveshown that cultured human keratinocytes switch between expandingand balanced modes of growth, which depend on cell-cell contactand EGF signaling (Roshan et al., 2016). Our findings support thismodel and identify matrix stiffness as a key upstream regulator. Inaddition, another recent study demonstrated that increased matrixstiffness promotes directional migration of HaCaT keratinocytes(Wickert et al., 2016). Together, these results and our own establisha mechanism for how keratinocytes sense and respond to changes inbulk material elasticity.Mechanistically, we show that EGF signaling mediates the

growth response of human keratinocytes to altered matrix stiffness,and phosphorylation of EGFR depends on focal adhesion assembly

and acto-myosin contractility. Moreover, this response involvesdirect interaction between EGFR and focal adhesions, suggestingthat focal adhesion signaling molecules, such as FAK, may regulateEGFR activity. However, it is also possible that EGFR activationregulates focal adhesion assembly, and potential bi-directionalsignaling and feedback mechanisms will be an important area offuture investigation. Our findings are consistent with previousstudies in normal and cancerous mammary epithelia (Klein et al.,2009; Paszek et al., 2005; Wang et al., 1998), as well as recentfindings, which link stiffness-dependent EGFR activation toSrc-family kinases in fibroblasts (Saxena et al., 2017). Thus,bio-mechanical regulation of focal adhesion assembly may play akey role in modulating EGF signaling across diverse cell types.

In addition to cell-matrix adhesions, cell-cell adhesions alsocontribute to the biomechanical regulation of EGFRs. In simple

Fig. 5. Focal adhesion assembly and cytoskeletal tension are required for stiffness-dependent changes in EGF signaling. (A,B) Representativefluorescence 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 whiletreated 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 aftertransfection with siRNA targeting PTK2 (FAK) or VCL, or with non-targeting control (NTC) siRNA. (E) Quantification of colony size (normalized to 20% DMSOlevel) 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.

6

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

Journal

ofCe

llScience

epithelia, increased substrate stiffness inhibits adherens junctionassembly, which in turn enhances EGF sensitivity and proliferation(Kim and Asthagiri, 2011). Recent studies also suggest that, in theepidermis, elevated acto-myosin tension within adherens junctionsin the granular layer negatively regulates EGFR activity (Rübsamet al., 2017). While our studies here aimed to establish the directsignaling between the ECM and EGFR by using sparse, low-Ca2+

culture conditions, the potential crosstalk with cell-cell adhesionmechanics cannot be completely excluded. It will be interesting infuturework to explore how biomechanical cues from both cell-ECMand cell-cell adhesions are integrated and regulate keratinocytefunction under more confluent, in vivo-like conditions. Directmeasurement of forces at cell-cell adhesions (Borghi et al., 2012)and localization of activated EGFR within different mechanicalenvironments will be of particular interest. Likewise, the role ofadditional cell adhesion receptors, such as desmosomes (Broussardet al., 2017), and other growth factor receptors (Conway et al., 2013)in cellular mechanosensing will be important areas of futureinvestigation. Finally, it will also be necessary to consider thedynamics of focal adhesion turnover and EGFR recycling, as EGFRis recycled together with β1 integrins (Caswell et al., 2008).Although many studies have investigated the effects of substrate

mechanics on cell function using controlled in vitromodels, only afew have linked in vivo biomechanics to normal or pathologicalfunctions (Gilbert et al., 2010; Levental et al., 2009). Our resultsindicate that EGFR phosphorylation correlates with increased

tissue stiffness in human skin and may play a role in keloid scarpathogenesis. While genome-wide association studies have onlylinked a handful of genes to keloid susceptibility (Nakashimaet al., 2010), the underlying causes of keloid scar formation remainalmost completely unknown. It is interesting to note that keloidsoften develop in areas of skin with high tension (Andrews et al.,2016; Huang et al., 2016; Ogawa et al., 2012), which combinedwith our findings, further supports a role for biomechanics in scarformation. Future studies examining the inter- and intra-keloidheterogeneity in mechanics, as well as the crosstalk with growthfactor signaling and epidermal cell mechanics, will be importantand will hopefully shed new light on these disfiguring and oftenpainful conditions.

MATERIALS AND METHODSSubstrate preparationPDMS substrates (Sylgard 184, Dow Corning) were prepared by mixingthe PDMS base with crosslinker at ratios varying from 50:1 (2%) to 5:1(20%). PDMSmixtures were de-gassed under a vacuum, spread onto 13 mmdiameter glass coverslips or 6-well plates, and cured overnight at 70°C. Tofunctionalize the PDMS substrates with ECM, the surfaces were coveredwith a solution of 50 mg/ml sulfo-SANPAH (Thermo Scientific) in waterand exposed to 365 nm UV light for 10 min. This process was repeatedtwice, followed by incubation with either 50 μg/ml rat type I collagen (BDBiosciences) or 50 μg/ml human plasma fibronectin. Samples were rinsedthree times with PBS, and sterilized with UVB and 70% ethanol prior to cellseeding. All chemicals were from Sigma-Aldrich unless otherwise noted.

Fig. 6. EGFR activation correlates with tissue stiffness in vivo. (A) Representative H&E staining for a keloid scar. The dotted circle indicates the approximatelocation within the dermis of the keloid edge where micro-indentation tests were performed. (B) Representative load-displacement curves for individualkeloid 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-keloidaffected donor; n=5 (normal) and =6 (keloid). *P<0.05. (D) Immunofluorescence staining for keratin 14 (K14) and pEGFR (Y1068) in frozen sections ofmatched keloid and extra-lesional skin. Scale bars: 100 µm. (E) Quantification of pEGFR fluorescence intensity for tissue sections stained for pEGFR Y1068 orpEGFR Y845. Data are expressed as mean fluorescence intensity within the basal layer of the epidermis of the keloid relative to the basal layer of theextra-lesional epidermis from the same patient (n=3 donors).

7

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

Journal

ofCe

llScience

Cell culturePrimary human keratinocytes were isolated from neonatal foreskin andmaintained on a layer of J2 3T3 fibroblasts in FAD medium as previouslydescribed (Rheinwald and Green, 1977). For studies on PDMS substrates,fibroblasts were removed using Versene (Invitrogen), and keratinocytes(passage 2–6) were trypsinized and seeded onto PDMS substrates inkeratinocyte serum-free medium (KSFM, Invitrogen) supplemented withbovine pituitary extract, penicillin/streptomycin, and 0.1 ng/ml EGF. Toinduce terminal differentiation, keratinocytes were cultured in KSFMfurther supplemented with 1.8 mM CaCl2.

Tissue samplesSkin samples were obtained from keloid patients and healthy volunteersfrom the plastic surgery department at Barts Health NHS Trust. All tissuesamples were from dark skinned (South Asian or Afro-Caribbean) adultdonors (male and female, under the age of 50); body sites included the back,shoulder, chest and stomach. All subjects gave informed consent and thestudy was conducted under local ethical committee approval (East LondonResearch Ethics Committee, study no 2011-000626-29).

Colony-formation assayPrimary keratinocytes were seeded onto non-ECM coated PDMS surfaceswithin a 6-well plate at a density of 1000/well. Cells were cultured for10 days in KSFM supplemented with 0–10 ng/ml EGF, 10 µM AG1478,50 µM Blebbistatin, or 416 nM FAK inhibitor 14 (Tocris Bioscience,Bristol, UK). Cells were fixed with 4% paraformaldehyde (PFA), stained for30 min with 0.06% Crystal Violet, and rinsed copiously with water.Scanned images of the stained wells were analyzed with ImageJ to quantifycolony size and number.

EdU labelingTo measure DNA synthesis, keratinocytes were cultured on PDMSsubstrates for 7 days in KSFM then incubated with 10 µM 5-ethynyl-2′-deoxyuridine (EdU) for 1 h at 37°C and rinsed twice with Edu-free KSFM.Cells were fixed with 4% PFA, and EdU incorporated into the DNA wastagged with AlexaFluor-568 by using the ‘Click-It’ kit (Thermo Scientific)according to the manufacturer’s instructions. Samples were co-stained withDAPI and imaged using a Leica DM5000B microscope.

Western blot analysisCells were washed in PBS and incubated in radioimmunoprecipitation assay(RIPA) buffer plus protease and phosphatase inhibitors for 10 min on ice.Cells were scraped off the dish, briefly sonicated, and centrifuged (300 g for5 min) to remove insoluble material. Protein concentration was determinedby the BCA assay (Thermo Scientific). Lysates were combined with loadingbuffer (Thermo Scientific) and 1% β2-mercaptoethanol, and equal amountsof total protein were resolved on a 4% or 10% polyacrylamide gel (Bio-Rad)and transferred onto nitrocellulose membranes (GE Lifesciences).Membranes were blocked for 1 h in either 5% non-fat dry milk or 3%BSA, before being incubated either 1 h at room temperature or overnight at4°C with primary antibodies against Involucrin [SY7, CRUK (1:1000)],pEGFRY1068 [cat. no. 3777, Cell Signaling Technology, Tyr1068 (D7A5)RbXP (1:1000)], EGFR [cat. no. 4267, Cell Signaling Technology (D38B1)Rb XP (1:1000)], pERK1/2 [cat. no. 9106S, Cell Signaling Technology(1:1000)], ERK1/2 [cat. no. 4695S, Cell Signaling Technology (1:1000)],pAkt [cat. no. 9271, Cell Signaling Technology Ser473 Rb (1:1000)], Akt[cat. no. 9272, Cell Signaling Technology Rb (1:1000)], Paxillin [cat. no.610569, BD Biosciences (1:1000)], pFAK [cat. no. 611722, BDBiosciences (1:500)], FAK [cat. no. 0537, Millipore (1:500)] or GAPDH[cat. no. ab9485, Abcam Rb (1:2000)]. Secondary detection was performedwith HRP-conjugated anti-rabbit or anti-mouse antibodies (1:5000, Dako).Proteins were visualized using the enhanced chemiluminescence detectionsystem (Millipore, Watford, UK).

ELISAThe enzyme-linked immunosorbent assay (ELISA) for amphiregulin wasperformed on conditioned medium from cells cultured on PDMS substratesfor 24 h according to the manufacturer’s protocol for the DuoSet ELISA

development kit (R&D Systems, Abingdon, UK). Briefly, ELISAMaxiSorp 96-well plates (Thermo Scientific) were coated overnight atroom temperature with the capture antibody diluted in a 1% BSA/PBSsolution. Plates were washed three times with 0.05% Tween 20 in PBS andblocked with 1% BSA for 1 h. Following a second wash step, 100 µl of theconditioned medium samples (pre-diluted 1:10) were incubated at roomtemperature for 2 h. The plate was washed three times, incubated with abiotinylated anti-human amphiregulin antibody (from the kit) for 2 h atroom temperature, and washed three more times. Detection was performedby incubation with 100 µl horseradish peroxidase (HRP)-taggedstreptavidin for 20 min, followed by washing and incubation with thesubstrate solution for 20 min. The reaction was stopped with 50 µl StopSolution and read at 450 nm using a Synergy HT plate reader.

Immunofluorescence and imagingFor immunofluorescence staining, keratinocytes on PDMS substrates werefixed with 4% PFA and permeabilized with 0.1% Triton X-100 for 5 min.Samples were blocked with 10% FBS plus 0.25% gelatin in PBS for 1 h andincubated overnight at 4°C with primary antibodies against pEGFR Y1068(as above, 1:500), pEGFR Y1086 [cat. no. ab32086, Abcam (1:500)],paxillin (as above, 1:500), vinculin [hVIN-1; cat. no. V9131, Sigma-Aldrich(1:1000)], E-cadherin [HECD1; cat. no. ab1416, Abcam (1:100)] or totalEGFR (as above, 1:100). Secondary staining of pEGFR antibodies wasperformed with anti-rabbit AlexaFluor-488 [cat. no A11008, ThermoScientific (1:1000)], and anti-mouse AlexaFluor-568 or-488 [cat. nosA10037 and A11001, Thermo Scientific (1:1000)]. F-actin was labeled withphalloidin-AlexaFluor-568 or -488 (1:500, Sigma-Aldrich) included in thesecondary solution. For the proximity ligation assay, fixed samples wereco-stained with antibodies against paxillin and EGFR, and detectedusing the Duolink Mouse-Rabbit Red kit (Sigma-Aldrich) according tothe manufacturer’s instructions. Samples weremounted on glass microscopeslides withMowiol and imaged using a Zeiss 710 confocal microscope (CarlZeiss) and 20× or 63× objectives.

Normal skin and keloid tissue samples were either snap frozen in optimalcutting temperature (OCT) compound (BD Biosciences) or fixed with 4%paraformaldehyde and embedded in paraffin. Freshly cut frozen sectionswere fixed for 10 min in 4% paraformaldehyde, blocked with 10% FBSand 0.25% gelatin, and stained with anti-pEGFR Y1068 (as above, 1:300).Paraffin sections were de-waxed, and heat-mediated antigen retrieval wasperformed with 10 mM sodium citrate. Sections were blocked as before andstained with anti-pEGFR Y845 (1:100, Cell Signaling, 6963S). Slides wereimaged with a Leica DM5000B microscope, and mean fluorescenceintensity of the basal layer was quantified with ImageJ.

siRNA knockdownKeratinocytes were seeded onto collagen-coated PDMS surfaces in a 6-wellplate and cultured overnight. Cells were transfected with 4 pmole siRNAand 4 µl of Jet Prime reagent (Polyplus Transfection) per well according tothe manufacturer’s instructions. Cells were cultured for 72 h and harvestedfor western blot analysis. Silencer Select (Thermo Fisher) validatedsmall interfering RNAs (siRNAs) were used for PTK2 (s11486) and VCL(s14764) knockdown.

Annexin V analysisApoptosis and viability were analyzed by flow cytometry. Keratinocyteswere cultured on PDMS substrates for 7 days, trypsinized, resuspended in200 µl Annexin V buffer (50 mMHEPES, 700 mMNaCl, 12.5 CaCl2) plus5 µl Annexin V-FITC (Invitrogen) and DAPI, and incubated at roomtemperature for 15 min. Annexin V-positive (apoptotic) and DAPI-positive(dead) cells were analyzed by flow cytometry using the Becton DickinsonLSRII. As a positive control for apoptosis, cells were treated with 10 mJ/cm2

UVB light 20 h prior to analysis.

Material characterization and mechanical testingFor the mechanical testing of the PDMS, samples were cast into Petri dishesand, after curing were cut to an approximate size of 5 mm wide×25 mmlong×1.5 mm thick. Tensile testing was conducted using an Instron

8

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

Journal

ofCe

llScience

universal testing system. The PDMS samples were held in place usingpneumatic grips and tested to failure at a strain rate of 20 mm/min. Theelastic modulus was calculated from the slope of the stress versus straincurve. Six samples per setting (2% and 20%) were tested.

For scanning electron microscopy (SEM) analysis of surface topography,cover slips were coated with PDMS and gold-coated using an Agar autosputter coater. SEM imaging was conducted using an FEI Inspect-F with anaccelerating voltage of 5 kV and a working distance of 10 mm. Theroughness measurements were performed using AFM (nTegra, NT-MDT).The samples were imaged over a 10×10 μm area in semi-contact mode witha silicon nitride cantilever (MLCT, Bruker, spring constant k=0.6 N/m). Thesample height at each point within the image and average roughness weremeasured using the AFM software (Nova, NT-MDT).

The tissue moduli of keloid and normal skin samples were measured bymicro-indentation. Frozen specimens were fully thawed at room temperaturein PBS for 1 h before testing. Mechanical indentation was performed usingan Instron ElectroPuls E1000 (Instron) equipped with a 10 N load cell(resolution=0.1 mN). Specimens were indented using a stainless steel plane-ended cylindrical punch with a diameter (Øi) of 2 or 1 mm. Specimenthickness (Ts) was measured as the distance between the base of the test dishand top of the sample, each detected by applying a pre-load of 0.3 mN.Specimen diameter (Øs) was measured using electronic callipers.Indentation was performed at room temperature with specimens fullysubmerged in PBS throughout testing. Tests were performed using a rampeddisplacement-control regime whereby each specimen was displaced to 30%of their measured thickness at a rate of 1% s−1. The resulting load detectedfrom the sample was recorded at 10 Hz. To minimise errors in calculationsof tissue moduli, specimen to indenter ratios were kept to Øs:Øi ≥4:1 andTs:Øi ≤2:1.

Tissuemoduli were calculated from the tangents of the final linear regionsof the load-displacement experimental data with the aid of a correctedmathematical model (Delaine-Smith et al., 2016). Briefly, the tissuemodulus (E) is related to the indentation contact stiffness (S) and the radiusof the flat punch indenter (a) by the following relationship:

E ¼ ½ðS=2akÞ=Gk� � ð1� n2Þ: ð1Þ

The geometric correction factor к accounts for large-deformation, non-linear behavior and values for strains >15% can be determined from linearinterpolation (Zhang et al., 1997). The second geometrical correction factor,Gк, is applied from Delaine-Smith et al. (2016). Poisson’s ratio (ν), wasassumed to be 0.499 for all specimens.

Statistical analysisAll data were analyzed by ANOVA and Tukey’s test for post-hoc analysiswith sample size of independent experiments or patients indicated in thefigure captions.

AcknowledgementsWe thank Dr Gary Warnes for assistance with annexin V analysis and Oscar Pundelfor maintenance of primary keratinocyte cultures.

Competing interestsFiona Kenny has carried out paid consultancy work for Metaphase Ltd.

Author contributionsConceptualization: J.T.C.; Formal analysis: F.N.K., R.D.-S., A.P.K.; Investigation:F.N.K., Z.D., R.D.-S., A.P.K., A.C.L., J.T.C.; Resources: Z.D., M.P.P.; Writing -original draft: F.N.K., J.T.C.; Writing - review & editing: J.T.C.; Supervision: M.M.K.,M.P.P., J.T.C.; Project administration: M.M.K.; Funding acquisition: J.T.C.

FundingThis work was funded by the Barts Charity (Large Grant 442/1032), the British SkinFoundation (PhD studentship grant no: 4052s for F.N.K.), and the EuropeanResearch Council (CANBUILD project 322566 for R.D.-S.).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.215780.supplemental

ReferencesAdams, J. C. andWatt, F. M. (1989). Fibronectin inhibits the terminal differentiation

of human keratinocytes. Nature 340, 307-309.Andrews, J. P., Marttala, J., Macarak, E., Rosenbloom, J. and Uitto, J. (2016).

Keloids: the paradigm of skin fibrosis-Pathomechanisms and treatment. MatrixBiol. 51, 37-46.

Assefa, Z., Garmyn, M., Bouillon, R., Merlevede, W., Vandenheede, J. R. andAgostinis, P. (1997). Differential stimulation of ERK and JNK activities byultraviolet B irradiation and epidermal growth factor in human keratinocytes.J. Invest. Dermatol. 108, 886-891.

Blanpain, C. and Fuchs, E. (2009). Epidermal homeostasis: a balancing act of stemcells in the skin. Nat. Rev. Mol. Cell Biol. 10, 207-217.

Borghi, N., Sorokina, M., Shcherbakova, O. G., Weis, W. I., Pruitt, B. L., Nelson,W. J. and Dunn, A. R. (2012). E-cadherin is under constitutive actomyosin-generated tension that is increased at cell–cell contacts upon externally appliedstretch. Proc. Natl. Acad. Sci. USA 109, 12568-12573.

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).The desmoplakin-intermediate filament linkage regulates cell mechanics. Mol.Biol. Cell 28, 3156-3164.

Bruckner-Tuderman, L., Mitsuhashi, Y., Schnyder, U. W. and Bruckner, P.(1989). Anchoring fibrils and type VII collagen are absent from skin in severerecessive dystrophic epidermolysis bullosa. J. Invest. Dermatol. 93, 3-9.

Caswell, P. T., Chan, M., Lindsay, A. J., McCaffrey, M. W., Boettiger, D. andNorman, J. C. (2008). Rab-coupling protein coordinates recycling of alpha5beta1integrin and EGFR1 to promote cell migration in 3D microenvironments. J. CellBiol. 183, 143-155.

Chowdhury, F., Li, Y., Poh, Y.-C., Yokohama-Tamaki, T., Wang, N. and Tanaka,T. S. (2010). Soft substrates promote homogeneous self-renewal of embryonicstem 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,W. T. S. and Watt, F. M. (2010). Actin and serum response factor transducephysical cues from the microenvironment to regulate epidermal stem cell fatedecisions. Nat. Cell Biol. 12, 711-718.

Conway, D. E., Breckenridge, M. T., Hinde, E., Gratton, E., Chen, C. S. andSchwartz, M. A. (2013). Fluid shear stress on endothelial cells modulatesmechanical tension across VE-cadherin and PECAM-1. Curr. Biol. 23,1024-1030.

Delaine-Smith, R. M., Burney, S., Balkwill, F. R. and Knight, M. M. (2016).Experimental validation of a flat punch indentation methodology calibrated againstunconfined compression tests for determination of soft tissue biomechanics.J. Mech. Behav. Biomed. Mater. 60, 401-415.

Gat, U., DasGupta, R., Degenstein, L. and Fuchs, E. (1998). De Novo hair folliclemorphogenesis and hair tumors in mice expressing a truncated beta-catenin inskin. Cell 95, 605-614.

Gilbert, P. M., Havenstrite, K. L., Magnusson, K. E. G., Sacco, A., Leonardi,N. A., Kraft, P., Nguyen, N. K., Thrun, S., Lutolf, M. P. and Blau, H. M. (2010).Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture.Science 329, 1078-1081.

Green, K. J. and Simpson, C. L. (2007). Desmosomes: new perspectives on aclassic. J. Invest. Dermatol. 127, 2499-2515.

Herrick, S. E., Sloan, P., McGurk, M., Freak, L., McCollum, C. N. and Ferguson,M. W. (1992). Sequential changes in histologic pattern and extracellular matrixdeposition during the healing of chronic venous ulcers. Am. J. Pathol. 141,1085-1095.

Huang, C., Liu, L., You, Z., Wang, B., Du, Y. and Ogawa, R. (2016). Keloidprogression: a stiffness gap hypothesis. Int. Wound J. 14, 764-771.

Jones, P. H. andWatt, F. M. (1993). Separation of human epidermal stem cells fromtransit amplifying cells on the basis of differences in integrin function andexpression. Cell 73, 713-724.

Jost, M., Kari, C. and Rodeck, U. (2000). The EGF receptor-an essential regulatorof multiple epidermal functions. Eur. J. Dermatol. 10, 505-510.

Kao, A. P., Connelly, J. T. and Barber, A. H. (2016). 3D nanomechanicalevaluations of dermal structures in skin. J. Mech. Behav. Biomed. Mater. 57,14-23.

Kim, J.-H. and Asthagiri, A. R. (2011). Matrix stiffening sensitizes epithelial cells toEGF and enables the loss of contact inhibition of proliferation. J. Cell. Sci. 124,1280-1287.

Klein, E. A., Yin, L., Kothapalli, D., Castagnino, P., Byfield, F. J., Xu, T., Levental,I., Hawthorne, E., Janmey, P. A. andAssoian, R. K. (2009). Cell-cycle control byphysiological matrix elasticity and in vivo tissue stiffening. Curr. Biol. 19,1511-1518.

Lee-Thedieck, C., Rauch, N., Fiammengo, R., Klein, G. and Spatz, J. P. (2012).Impact of substrate elasticity on human hematopoietic stem and progenitor celladhesion and motility. J. Cell Sci. 125, 3765-3775.

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 crosslinkingforces tumor progression by enhancing integrin signaling. Cell 139, 891-906.

Martins, V. L., Vyas, J. J., Chen, M., Purdie, K., Mein, C. A., South, A. P., Storey,A., McGrath, J. A. and O’Toole, E. A. (2009). Increased invasive behaviour in

9

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

Journal

ofCe

llScience

cutaneous squamous cell carcinoma with loss of basement-membrane type VIIcollagen. J. Cell. Sci. 122, 1788-1799.

Nakashima, M., Chung, S., Takahashi, A., Kamatani, N., Kawaguchi, T.,Tsunoda, T., Hosono, N., Kubo, M., Nakamura, Y. and Zembutsu, H. (2010).A genome-wide association study identifies four susceptibility loci for keloid in theJapanese population. Nat. Genet. 42, 768-771.

Niessen, C. M. (2007). Tight junctions/adherens junctions: basic structure andfunction. J. Invest. Dermatol. 127, 2525-2532.

Ogawa, R., Okai, K., Tokumura, F., Mori, K., Ohmori, Y., Huang, C., Hyakusoku,H. and Akaishi, S. (2012). The relationship between skin stretching/contractionand pathologic scarring: the important role of mechanical forces in keloidgeneration. Wound Repair. Regen. 20, 149-157.

Paszek, M. J., Zahir, N., Johnson, K. R., Lakins, J. N., Rozenberg, G. I., Gefen, A.,Reinhart-King, C. A., Margulies, S. S., Dembo, M., Boettiger, D. et al. (2005).Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241-254.

Piepkorn, M. (1996). Overexpression of amphiregulin, a major autocrine growthfactor for cultured human keratinocytes, in hyperproliferative skin diseases.Am. J. Dermatopathol. 18, 165-171.

Piepkorn, M., Lo, C. and Plowman, G. (1994). Amphiregulin-dependentproliferation of cultured human keratinocytes: autocrine growth, the effects ofexogenous recombinant cytokine, and apparent requirement for heparin-likeglycosaminoglycans. J. Cell. Physiol. 159, 114-120.

Reiss, M. and Sartorelli, A. C. (1987). Regulation of growth and differentiation ofhuman keratinocytes by type beta transforming growth factor and epidermalgrowth factor. Cancer Res. 47, 6705-6709.

Rheinwald, J. G. and Green, H. (1977). Epidermal growth factor and themultiplication of cultured human epidermal keratinocytes. Nature 265, 421-424.

Roshan, A., Murai, K., Fowler, J., Simons, B. D., Nikolaidou-Neokosmidou, V.and Jones, P. H. (2016). Human keratinocytes have two interconvertible modesof proliferation. Nat. Cell Biol. 18, 145-156.

Rubsam, M., Mertz, A. F., Kubo, A., Marg, S., Jungst, C., Goranci-Buzhala, G.,Schauss, A. C., Horsley, V., Dufresne, E. R., Moser, M. et al. (2017). E-cadherinintegrates mechanotransduction and EGFR signaling to control junctional tissuepolarization and tight junction positioning. Nat. Commun. 8, 1250.

Saxena, M., Liu, S., Yang, B., Hajal, C., Changede, R., Hu, J., Wolfenson, H.,Hone, J. and Sheetz, M. P. (2017). EGFR andHER2 activate rigidity sensing onlyon rigid matrices. Nat. Mater. 16, 775-781.

Stojadinovic, O., Brem, H., Vouthounis, C., Lee, B., Fallon, J., Stallcup, M.,Merchant, A., Galiano, R. D. and Tomic-Canic, M. (2005). Molecularpathogenesis of chronic wounds: the role of beta-catenin and c-myc in theinhibition of epithelialization and wound healing. Am. J. Pathol. 167, 59-69.

Trappmann, B., Gautrot, J. E., Connelly, J. T., Strange, D. G., Li, Y., Oyen, M. L.,Cohen Stuart, M. A., Boehm, H., Li, B., Vogel, V. et al. (2011). Extracellular-matrix tethering regulates stem-cell fate. Nat. Mater. 11, 642-649.

Uribe, P. and Gonzalez, S. (2011). Epidermal growth factor receptor (EGFR) andsquamous cell carcinoma of the skin: molecular bases for EGFR-targetedtherapy. Pathol. Res. Pract. 207, 337-342.

Wan, Y. S., Wang, Z. Q., Shao, Y., Voorhees, J. J. and Fisher, G. J. (2001).Ultraviolet irradiation activates PI 3-kinase/AKT survival pathway via EGFreceptors in human skin in vivo. Int. J. Oncol. 18, 461-466.

Wang, F., Weaver, V. M., Petersen, O. W., Larabell, C. A., Dedhar, S., Briand, P.,Lupu, R. and Bissell, M. J. (1998). Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basementmembrane breast cultures: a different perspective in epithelial biology. Proc. Natl.Acad. Sci. USA 95, 14821-14826.

Wickert, L. E., Pomerenke, S., Mitchell, I., Masters, K. S. and Kreeger, P. K.(2016). Hierarchy of cellular decisions in collective behavior: implications forwound healing. Sci. Rep. 6, 20139.

Wysocki, A. B., Staiano-Coico, L. and Grinnell, F. (1993). Wound fluid fromchronic leg ulcers contains elevated levels of metalloproteinases MMP-2 andMMP-9. J. Invest. Dermatol. 101, 64-68.

Zhang, M., Zheng, Y. P. and Mak, A. F. T. (1997). Estimating the effective Young’smodulus of soft tissues from indentation tests–nonlinear finite element analysis ofeffects of friction and large deformation. Med. Eng. Phys. 19, 512-517.

Zhu, A. J. and Watt, F. M. (1999). beta-catenin signalling modulates proliferativepotential of human epidermal keratinocytes independently of intercellularadhesion. Development 126, 2285-2298.

10

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

Journal

ofCe

llScience