Polydimethylsiloxane (PDMS) substrates (Sylgard 184, Dow Corning Corp., USA) were prepared by mixing the PDMS base with a crosslinker at a ratio of 50:1 to 10:1. PDMS mixtures were degassed under vacuum, spread onto 13 mm diameter glass coverslips or 6-well plates, and cured overnight at 70°C. To functionalize the PDMS substrates with ECM, the surfaces were covered with a solution of 50 mg/mL sulfo-SANPAH (Thermo Fisher Scientific, USA) in water and exposed to 365 nm UV light for 10 min. This process was repeated twice, followed by incubation with 50 μg/mL human plasma fibronectin. The samples were rinsed three times with PBS and sterilized with UVB and 70% ethanol prior to cell seeding. All chemicals were purchased from Sigma-Aldrich (St. Louis, MI) unless otherwise noted.

The mechanical properties of the PDMS substrates were measured using a custom-made indenter consisting of a load cell and an automated stage (22–24). A spherical tip with a diameter of 5 mm was used for the indentation. Young’s modulus of PDMS at various crosslinker concentrations were estimated by fitting an indentation force-depth curve to the Hertz contact model.

The human keratinocyte cell line HaCaT was provided by the German Cancer Research Center (Heidelberg, Germany). Normal human epidermal keratinocytes (NHEK) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). HaCaT cells were cultured in Dulbecco’s modified Eagle medium, which was supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA), 1% penicillin–streptomycin (Invitrogen), and 1% non-essential amino acids (Gibco, Thermo Fisher Scientific, USA). NHEK cells were cultured in EpiLife serum-free medium containing EpiLife undefined growth supplement (Thermo Fisher Scientific). The cells were cultured at 37°C in a humidified incubator containing 5% CO2.

Cell viability was detected using the cell counting-kit-8 assay (Dojindo, Kumamoto, Japan). HaCaT cells were seeded in a 96-well plate with varying elastic moduli (low, medium, and high stiffness) at a density of 1.2 × 10⁵ cells/mL (100 μL total volume/well). At 24, 48, 72, and 96 h after incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 4 h at 37°C, in accordance with the manufacturer’s instructions. The OD at 450 nm was measured using a VICTORTM X3 microplate reader (Perkin-Elmer, Waltham, MA, USA).

Chemicals 2,4-dinitrochlorobenzene (DNCB, Sigma-Aldrich) and lactic acid (Sigma-Aldrich) were dissolved in dimethyl sulfoxide (the maximum concentration of DMSO in the culture medium was 0.1%) and PBS respectively. For in vitro treatment, the HaCaT cells cultured on PDMS substrates were treated with 5 μg/mL DNCB or 1 mg/mL lactic acid (Sigma-Aldrich) for 24 h. The culture medium supplemented with 0.1% DMSO or PBS was used as a vehicle control. The concentrations of DNCB and lactic acid used in this study were determined based on prior tests of 75% cell viability (CV75), as described previously (20).

Cells were lysed in RIPA buffer containing a protease inhibitor and phosphatase inhibitor (GenDepot, Houston, TX, USA). The protein samples were separated on a 12% SDS-PAGE gel. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), washed with PBS containing 0.05% Tween-20 (PBST), and blocked with PBS containing 5% non-fat dry milk for 1 h at room temperature. The membranes were then incubated overnight at 4°C with primary antibodies against β1 integrin (#4706, 1:1000, Cell Signaling Technology, Danvers, MA, USA), phospho-ERK1/2 (#5726, 1:1000, Cell Signaling Technology), total ERK1/2 (#4695, 1:1000, Cell Signaling Technology), phospho-PI3K (#3087, 1:1000, Cell Signaling Technology), total PI3K (#3087, 1:1000, Cell Signaling Technology), phospho-Akt (#9271, 1:1000, Cell Signaling Technology), total Akt (#9272, 1:1000, Cell Signaling Technology), and β-actin (sc-47778, 1:3000, Santa Cruz Biotechnology, USA). The following day, the membranes were washed three times with PBST and further incubated for 1 h at room temperature with the appropriate HRP-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology). Proteins were visualized using an enhanced chemiluminescence light-detecting kit (34095, SuperSignal West Femto, Thermo Fisher Scientific).

For immunofluorescence staining, keratinocytes on PDMS substrates were fixed in 4% paraformaldehyde (Electron Microscopy Science) and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich). For actin and nucleus staining, the cells were incubated with Alexa-488-labeled phalloidin (Invitrogen) and Hoechst 33342 (Sigma-Aldrich) in 1% BSA (Sigma-Aldrich) blocking solution. For focal adhesion staining, the cells were incubated with a focal adhesion kinase (Proteintech, Rosemont, IL, USA) primary antibody for 1 h at room temperature. After washing, the cells were incubated for 1 h with Alexa-568-labeled goat anti-rabbit secondary antibody (Thermo Fisher Scientific). All images were obtained using an upright microscope (Nikon) with Plan Fluor 20× (NA 0.5) and S Plan Fluor 40× (NA 0.6) objectives.

After 24 h of incubation under chemical or mechanical stimulation, as described previously (2, 21), ELISA for IL-6 was performed on conditioned media from cells on PDMS substrates, in accordance with the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA).

For quantitative real-time PCR analysis, total RNA was solubilized in TRIzol reagent (Invitrogen) and extracted according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using reverse transcription, and the amount of mRNA was determined using real-time PCR analysis with the SYBR Green qPCR premix (Enzynomics, Daejeon, South Korea) on an ABI real-time PCR 7500 machine (Applied Biosystems, CA, USA). The primer sequences were as follows: human IL-6 forward, 5-AAATTCGGTACATCCTCGAC-3; human IL-6 reverse, 5-CAGGAACTGGATCAGGACTT-3; human β-actin forward, 5-ATTGCCGACAGGATGCAGAA-3; and human β-actin reverse, 5 -GCTGATCCACATCTGCTGGAA-3.

All statistical analyses were conducted using the GraphPad Prism software (version 8.0) and displayed as the mean ± standard error of the mean (SEM). A p-value less than 0.1 was considered to indicate a significant difference (*p < 0.1; **p < 0.01; ***p < 0.001; and ****p < 0.0001).

To investigate the effect of matrix stiffness on keratinocytes, we employed a cell culturing system using PDMS with low (20 kPa), medium (500 kPa), or high (1200 kPa) elastic modulus ( Supplementary Figure S1 ). Although there is discrepancy in the references, the elastic modulus of the normal skin is 20~140kPa and the modulus further increases during either normal physiological formation of mature granulation tissue after injury (200kPa~) or pathological excessive ECM production in the skin with stiff keloid scar tissue (100~1000 kPa) (12, 25, 26). Thus, 20 kPa substrate mimicked the stiffness of normal skin, and the substrates with 500 and 1200 kPa were most similar to mature granulation tissues and pathological skin diseases respectively.

First, we cultured HaCaT cells on PDMS substrates with varying elastic moduli for 4 days and observed the influence of matrix stiffness on long-term keratinocyte growth. Cell growth tracking revealed a more rapid increase in the number of cells on the stiff substrate from day 2, and the difference gradually increased over the 4 days, which is consistent with the results of previous studies (10, 12) ( Supplementary Figure S2 ).

To assess whether the alteration of matrix stiffness causes cytoskeletal remodeling, cell morphology and cytoskeletal organization were evaluated in keratinocytes cultured on substrates with low (20 kPa) or high (1200 kPa) stiffness. Consistent with the results presented in Supplementary Figure S2 , there were no detectable differences in the number of keratinocytes on day 1; however, the cells cultured on the stiffer substrate (high) were well spread with visible actin stress fibers compared to those on the soft substrate (low) ( Figure 1A ). Additionally, quantification analysis using Cell Profiler (21) showed that the adhesive contact area of cells and nuclei increased dramatically with increasing substrate stiffness ( Figure 1B ). A cell or nucleus was fitted to an ellipse. The aspect ratio (AR) of cell or nucleus was estimated by dividing the long axis by short one of the ellipse (27, 28). There were no significant differences in terms of AR and orientation of cells and nuclei over the range of matrix moduli ( Figures 1C–E ). Collectively, these results indicate that the actin cytoskeleton and nucleus structurally and biochemically adapt to changes in the matrix substrate, which is responsible for transducing diverse mechanical signals into cellular responses.

Considering the function of the actin cytoskeleton and nucleus in cellular mechanotransduction to modulate gene expression (29–32), we next investigated whether alteration of the substrate matrix could concurrently influence pro-inflammatory cytokine production. To address this question, we compared the stiffness-mediated production of the proinflammatory cytokine IL-6, which has been shown to be increased in keratinocytes by mechanical stretching coupled with chemical stimulation in our previous study (21) ( Figure 2A ). Without no chemical stimulation, we observed an indistinguishable difference in IL-6 mRNA expression ( Figure 2B ) between keratinocytes cultured on substrates with varying stiffness. However, when DNCB with strong sensitizing potential was added to the cells, IL-6 expression was significantly enhanced with increasing substrate stiffness in keratinocytes. Notably, unlike keratinocytes treated with DNCB, the observed stiffness-mediated IL-6 production was not shown in response to the non-sensitizer lactic acid (LA), suggesting that mechanical cues in concert with strong chemical stimulation are required for IL-6 production. The results showing that increased substrate stiffness stimulated IL-6 in response to DNCB were consistent with those observed at the protein level, as measured using ELISA ( Figure 2C ). A similar higher increase in the level of IL-6 production was found in NHEK cells cultured on the stiffer substrate ( Supplementary Figure S3 ). Collectively, we conclude that increased matrix stiffness promotes IL-6 production in keratinocytes upon treatment with chemicals with strong skin sensitizing potential to evoke an inflammatory response.

Focal adhesions play an essential role in integrating mechanical properties of the extracellular matrix into biochemical and transcriptional responses, thereby regulating cell behaviors, including cell adhesion, migration, and differentiation (33, 34). To gain insight into the mechanisms underlying matrix stiffness-mediated IL-6 production in response to DNCB, we first examined the focal adhesion detected by FAK, a central signaling hub between integrin and multiple downstream cellular signaling pathways (35, 36) in keratinocytes cultured on substrates of different stiffness. FAK immunofluorescence imaging revealed that the cells established more adhesive interactions, as evidenced by both the area and length of FAK at the cell periphery, along with more visible stress fibers on the stiffer substrate (high) than on the soft substrate (low) ( Figures 3A, B ). Importantly, the difference in FAK activation between the substrates was more significant after DNCB treatment, further supporting a possible role of FAK in regulating stiffness-mediated IL-6 production ( Figures 3C–F ).

Focal adhesion transduces mechanical cues from the altered ECM to regulate cell behavior, in which integrins act as a coupler of mechanotransducers to initiate biochemical signaling (37, 38). In particular, recent studies have suggested that increased matrix-stiffness-dependent β1 integrin expression and clustering promote focal adhesions and subsequent mechanotransduction pathways including FAK-ERK and FAK-PI3K/Akt, which are involved in modulating the pathogenesis of various diseases (38–41). Furthermore, it is notable that β1 integrin-PI3K/Akt signaling pathway is involved in the upregulation of cytokine VEGF in cancer cells (41). Thus, we focused on the β1 integrin and its downstream ERK and PI3K/Akt signaling as possible mechanisms underlying matrix stiffening-mediated IL-6 secretion in keratinocytes.

Immunoblot analyses showed that β1 integrin was enhanced with increasing substrate stiffness independent of chemical stimuli ( Figures 4A, B ). Additionally, we observed that matrix stiffening induced phosphorylation of ERK and PI3K, followed by phosphorylation of Akt, as possibly subsequent downstream pathways of β1 integrin activation ( Figures 4C–E ). When DNCB was treated, the difference was more evident. These observations indicate that β1 integrin, ERK and PI3K/Akt pathways, which were activated with increasing matrix, were upregulated in keratinocytes exposed to DNCB.

We next investigated whether blocking ERK and PI3K/Akt pathways prevents matrix stiffness-dependent enhanced production of IL-6. To this end, we inhibited the ERK or PI3K/Akt pathway with PD98059 (ERK inhibitor) or LY294002 (PI3K/Akt inhibitor) and observed an overall decrease in IL-6 production in DNCB-treated keratinocytes ( Figure 4F ). Moreover, upon treatment with PD98059 or LY294002, IL-6 production in keratinocytes on the substrates with the highest stiffness was reduced to levels comparable to those on the least stiff substrate. Co-inhibition of ERK and PI3K/Akt with combination treatment completely abolished matrix-stiffening-enhanced IL-6 production in keratinocytes. Collectively, these results demonstrate that matrix-stiffening-mediated signal transduction through activation of ERK or PI3K/Akt pathway cooperates with chemical stimuli to drive IL-6 production.