The epidermis functions as a barrier to protect against chemical, biological, and physical insults. It is primarily composed of keratinocytes (McGrath et al., 2008) which alter their physical properties as they stratify from the basement membrane towards the external environment. This process culminates in the formation of the tough and waterproof outer layer of the skin (Simpson et al., 2011). Basal keratinocytes are the only layer of the epidermis in contact with the extracellular matrix (ECM) at the dermal-epidermal junction and must therefore undergo a phenotypic switch to enable rapid collective cell migration (CCM) to reinstate the skin barrier following injury (Haeger et al., 2015).

During CCM, ‘leader’ cells at the free edge sense the external environment. This activity requires cells adjacent to the injury site to undergo partial epithelial–mesenchymal transition (EMT), to reorganize the cytoskeleton and facilitate a migratory phenotype. Leader cells also relay signals to the attached ‘follower’ cells behind the free edge. This action coordinates speed and the direction of CCM. Sustained intercellular adhesions are essential to this process, to maintain both cell polarity and contacts with neighboring cells for organized movement (Li et al., 2012). Basal keratinocytes must also undergo proliferation following injury to replenish lost cells. However, proliferation can impede migration, as the cell must detach to round and divide. Therefore, the spatio-temporal co-ordination of keratinocyte migration and proliferation is critical for effective wound closure following injury; proliferation is restricted to a region away from the migratory front, enabling keratinocytes to reinstate the population without impeding migration speed (Park et al., 2017). CCM therefore requires coordination of cell–cell and cell–ECM adhesions to maintain epidermal integrity, organize the cell cytoskeleton, and regulate signaling pathways to promote appropriate zonal control of migration and proliferation.

Integrins are the primary family of cell surface receptors to facilitate basal keratinocyte attachment to the ECM. Integrins are non-covalently attached heterodimers formed of one α and one β subunit. There are 24 known α/β combinations in mammals, created by 18 α and eight β subunits (Hynes 2002). Integrins sense ECM through the binding of short amino acid recognition sequences e.g., the RGD motif in fibronectin and laminin. ECM is bound at the cleft between integrin headpieces. Therefore, the heterodimer combination determines ligand-binding specificity. Following ECM attachment, integrins couple the ECM to the F-actin cytoskeleton to generate traction (Case and Waterman 2015). The actin cytoskeleton provides the intrinsic force to propel the cell and to control shape change during migration (Svitkina 2018). However, integrins contain no intrinsic actin binding domain or kinase activity, and therefore facilitate assembly of multi-protein intracellular adhesion complexes called focal adhesions (FA) to modulate spreading and migration (Zaidel-Bar et al., 2007; Horton et al., 2015). The proteins recruited to FA can also modulate longer-term events such as survival, growth, and proliferation through intracellular signaling pathways (reviewed in (Vachon 2011)). Therefore, integrins are important in coordinating leader and follower cell dynamics during CCM.

α4 and α9 integrins are poorly described in the context of skin biology when compared to the other α integrin mammalian subunits. However, α4 and α9 integrins have both previously been associated with epithelial tissue repair; α4 and α9 integrins bind to components of the provisional ECM (Yokosaki et al., 1999; Greena et al., 2001; Liao et al., 2002), and the expression level of both integrins increases following injury (Häkkinen et al., 2000; Singh et al., 2004; Chung et al., 2018). Furthermore, α4 and α9 integrins are known to promote a migratory phenotype during epidermal injury repair (Liao et al., 2002; Singh et al., 2004). Similarly, studies of migrating mesenchymal cells demonstrate that both α4 and α9 integrins regulate lamellipodia actin polymerization and maintain front-rear polarity, both of which are important during CCM (Hight-Warburton and Parsons 2019). These data suggest that α4 and α9 integrins may regulate processes associated with the migratory phase of skin repair following injury. However, previous work in epithelia has only defined the expression and localization of these integrins at the tissue scale (Singh et al., 2004; Chung et al., 2018). As such, α4 and α9 integrin cytoplasmic binding partners and subcellular localization in keratinocytes are currently unknown.

Here, we demonstrate that α4 and α9 integrins show distinct subcellular localization under homeostatic conditions but undergo re-localization to co-localize upon wounding. This redistribution promotes keratinocyte CCM. We further demonstrate that α4 and α9 integrins share common intracellular binding partners that are required to suppress local activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Mitogen and Stress activated kinase 1 (MSK1) in keratinocyte monolayers. This signaling leads to coordinated actin protrusion and efficient directed migration. Our data therefore provide new insight into α4 and α9 integrin functions in epithelial homeostasis and repair.

Antibodies: Primary antibodies used were ERK1/2 (Cell Signaling Technology, Danvers, MA, United States), GAPDH (GeneTex, Irvine, CA, United States), GFP (MBL International Corporation, Woburn, MA, United States), Heat shock cognate 71 kDa protein (Sigma-Aldrich, St. Louis, MO, United States), Importin 7 (Abcam, Cambridge, United Kingdom), Integrin α2 (Thermo Fisher Scientific, Waltham, MA, United States), Integrin α4 Western blotting (NBP1-77333, Novus Biologicals, Centennial, CO, United States), Integrin α4 immunofluorescence (NBP1-77333, Novus Biologicals or Clone # 7.2R, R&D Systems, Inc.), Integrin α9 immunofluorescence (NBP2-16972, Novus Biologicals or Clone #Y9A2, Abcam), α9 integrin Western blotting (Clone # 3E4, Abnova, Taipei, Taiwan), α9 integrin function blocking (Clone #Y9A2, Bio-Rad Laboratories, Inc. Hercules, CA, United States), Integrin β1 (AB 1952, Merck Millipore, Burlington, MA, United States), Integrin β1 active (Clone # 12G10, Santa Cruz Biotechnology, Inc. CA, United States), MSK1 (R&D Systems, Inc. MN, United States), p53 (Cell Signaling Technology), Paxillin (Thermo Fisher Scientific and BD Bioscience, Franklin Lakes, NJ, United States), T202/Y204 phosphorylated ERK1/2 (Cell Signaling Technology), S380/S386/S377 phosphorylated RSK1/2/3 (R&D Systems, Inc.), S376/S360 phosphorylated MSK1/2 (R&D Systems, Inc.), RSK1 (R&D Systems, Inc.), Talin (Sigma-Aldrich), TNFR1 (Cell Signaling Technology), anti-species Horseradish Peroxidase (HRP)‒conjugated secondary antibodies (Dako, Glostrup, Denmark), AlexaFluor-conjugated antibodies (Thermo Fisher Scientific), Phalloidin (Thermo Fisher Scientific), and Hoechst (Sigma-Aldrich).

CDNA constructs: Integrin alpha4 EGFP-N3 was used, as described in (Parsons et al., 2008). Integrin alpha9 EGFP-N3 was a gift from Dean Sheppard (Addgene plasmid #13600). To generate integrin α9-TagRFP, a full-length clone of human α9 integrin in EGFP-N3 was used as a backbone (Addgene plasmid #13600). For the generation of α9-TagRFP, the coding region of the TagRFP was removed from TNFR1-TagRFP (Morton et al., 2019) using Kpn1 at the 3′ end and Not1 at the 5′ end. These sites were used to subclone TagRFP into EGFP cassette to give α9-pTagRFP-N3. Phusion cloning was carried out using Phusion® High-Fidelity PCR Master Mix with HF Buffer.

Tenascin-C and Tenascin-C (RAA) production: Glutathione S-transferase fusion proteins containing either wild type Tenascin-C or variant Tenascin-C (RAA) (a kind gift from Yasuyuki Yokosaki) were prepared by E.coli expression of the pGEX plasmids (Yokosaki et al., 1994); transformed bacteria were incubated until A ₆₀₀ reached 0.3–5 at 30 °C, at which time isopropyl-1-thio-β-galactopyranoside was added to a final concentration of 0.1 mM. Cultures were grown for several more hours, before cells were collected and sonicated (Sonics VC70T, 2 min with 10 s pulses at 15% amplitude). Glutathione S-transferase fusion proteins were affinity-purified with glutathione-Sepharose 4B beads (GE Healthcare, Chicago, IL, United States) and then cleaved off from glutathione S-transferase with PreScission Protease (GE Healthcare).

Cell culture: HPV16 immortalized human keratinocytes (NEB-1) (Morley et al., 1995) were maintained in RM + media (Fahmy et al., 1993), composed of high glucose Dulbeccos modified eagle medium (DMEM, Sigma-Aldrich) supplemented with 30% (v/v) Ham’s F12 Nutrient Mixture (Sigma-Aldrich), 10% (v/v) Fetal Bovine Serum (Thermo Fisher Scientific), 2 mM l-Glutamine (Sigma-Aldrich), 100 unit/mL penicillin and 0.1 mg/ml streptomycin (Sigma-Aldrich), 1x RM + containing 40 μg/ml hydrocortisone (Sigma-Aldrich), 500 μg/ml insulin (Sigma-Aldrich), 1 μg/ml EGF (PeproTech, Rocky Hill, NJ, United States), 0.84 μg/ml cholera toxin (Sigma-Aldrich), 500 μg/ml transferrin (Sigma-Aldrich) and 1.3 μg/ml Lyothyronine (Sigma-Aldrich). Keratinocytes were maintained at 37 °C in a 5% CO₂ humidified atmosphere.

CDNA Transfection: Nucleoporation was used to transfect keratinocytes with cDNA. Briefly, 5 × 10⁶ cells were resuspended in 100 µl of Nucleofector® Solution (Lonza, Basel, Switzerland). Plasmids were added to cell suspension, mixed, and then transferred to a cuvette. Nucleofector® Program U-020 (Amaxa Nucleofector® II 20800900, Lonza) was used to transfect the cells.

SIRNA transfection: Keratinocytes were transfected with either a siRNA pool targeting MSK1 (GE Healthcare Dharmacon, Inc. Lafayette, CO, United States) or a non-targeting control siRNA pool (GE Healthcare Dharmacon, Inc.). Cells were incubated for 6–8 h at 37°C with the transfection solution containing siRNA and DharmaFECT (GE Healthcare Dharmacon, Inc.) in Opti-MEM (Thermo Fisher Scientific) before the media was aspirated and replaced with normal growth media. Cells were incubated for 48 h prior to experimental use.

Treatments: To inhibit α4 and α9 integrins simultaneously, keratinocytes were treated with 5 μM N-(Benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl) tyrosine sodium salt (BOP, Tocris, Bristol, United Kingdom) or 1 µM LDV peptide (Tocris) for up to 48 h 5µM methyl-BOP (MBOP, Tocris) was used as an inactive BOP control in these experiments. To inhibit α4 integrin alone, keratinocytes were treated with 10 µM BIO1211 (Tocris) for up to 48 h. To track internalized α4 integrin with time-lapse microscopy, keratinocyte monolayers transiently expressing GFP tagged α4 integrin were treated with 5 µM BOP conjugated to Janelia Fluor® 646 (BOP-JF646, Tocris). To ascertain integrin stability, keratinocytes were treated with 10 μg/ml cycloheximide (Sigma-Aldrich), 100 μM Leupeptin (Bachem Holding AG, Bubendorf, Switzerland), and 20 μM MG132 (Tocris) for up to 48 h. To inhibit ERK1/2 signaling, keratinocytes were treated with 10 μM U0126 (Tocris) for up to 1 h. To inhibit MSK1, keratinocytes were treated with 5 µM RMM46 (Tocris) for up to 24 h. To activate α9 integrin, keratinocytes were treated with 10 μg/ml Tenascin-C (RAA) for up to 4 h, and 10 μg/ml Tenascin-C was used as a control in these experiments. To inhibit importin activity, keratinocytes were treated with 25 µM Ivermectin (Sigma-Aldrich), or 50 µM Importazole (Cayman Chemical, Michigan, United States) for up to 1 h.

Fluorescence Polarization: Measurements were performed on a BMG Labtech PolarStarOmega plate reader (BMG LABTECH Ltd., Buckinghamshire, United Kingdom) at 27°C by incubating 60 nM BOP conjugated to Janelia Fluor® 549 (BOP-JF549, Tocris) with the indicated integrins (Bio-Techne) in the concentration range 0–500 nM in PBS, pH 7.4 supplemented with 1 mM MgCl₂ and 1 mM CaCl₂. Estimation of equilibrium dissociation constant (K _D) values was performed assuming a one-site specific-binding model using the Prism package (GraphPad Software, San Diego, CA, United States). Data points are the mean of three replicates.

GFP-TRAP® Immunoprecipitation: Keratinocytes transiently expressing GFP-tag proteins were lysed in cold GFP TRAP lysis buffer (50 mM Tris (pH 7.4) (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), 1% NP40 (Sigma-Aldrich), 15 mM MgCl₂ (Sigma-Aldrich), 10% glycerol (Sigma-Aldrich), 1% Triton X-100 (Thermo Fisher Scientific), 5 mM EDTA (Sigma-Aldrich), Protease inhibitor cocktail set 1 (Merck Millipore)) was added to the cells. After a 10-min incubation on ice, keratinocytes were scraped and centrifuged for 10 min at 4°C. Supernatant was then incubated with 1:1 of GFP-TRAP® A beads (Chromotek, Planegg-Martinsried, Munich, Germany) and control agarose resin (Thermo Fisher Scientific) for 2 hours. Beads were then washed and 2x sample buffer (60 mM Tris-HCl (pH 6.8) (Sigma-Aldrich), 25% Glycerol (Sigma-Aldrich), 2.5% SDS (Sigma-Aldrich), 0.02% Bromophenol blue (Sigma-Aldrich), 2% β-mercaptoethanol (Sigma-Aldrich)) was added to the beads. Samples were boiled for 5 minutes at 95°C, and then subjected to either silver stain or Western blot analysis.

Liquid chromatography with tandem mass spectrometry analysis (LC-MS/MS): GFP-TRAP complexes were fully resolved on a SDS PAGE gel. Bands of interest were excised and incubated with 10 mM dithiothreitol at 56°C and then alkylated with 55 mM iodoacetamide at room temperature. Samples were digested using 1:20 (enzyme:substrate) ratio of bovine trypsin incubated in a shaking heat block at 37°C for 16 h. Peptides were extracted with aqueous dehydration/hydration using acetonitrile and 50 mM triethylammonium bicarbonate, pooled and dried. Samples were resuspended in 2% (v/v) acetonitrile, 0.05% (v/v) formic acid and peptides were resolved by reversed phase chromatography on a 75 μm C18 Pepmap column (50 cm length) using a three-step linear gradient of 80% acetonitrile in 0.1% formic acid (U3000 UHPLC NanoLC system; ThermoFisherScientific, United Kingdom). The gradient was delivered to elute the peptides at a flow rate of 250 nl/min over 60 min starting at 5% B (0–5 min) and increasing solvent to 40% B (5–40 min) prior to a wash step at 99% B (40–45 min) followed by an equilibration step at 5% B (45–60 min). The eluate was ionized by electrospray ionization using an Orbitrap Fusion Lumos (ThermoFisherScientific, United Kingdom) operating under Xcalibur v4.1.5. The instrument was programmed to acquire in automated data-dependent switching mode, selecting precursor ions based on their intensity for sequencing by collision-induced fragmentation using an Orbitrap-Ion Trap method defining a 3s cycle time between a full MS scan and MS/MS fragmentation. Orbitrap spectra (FTMS1) were collected at a resolution of 120,000 over a scan range of m/z 375–1,500 with an automatic gain control (AGC) setting of 4.0e5 with a maximum injection time of 35 ms. Monoisotopic precursor ions were filtered using charge state (+2–+7) with an intensity threshold set between 5.0e3–1.0e20 and a dynamic exclusion window of 35s ±10 ppm. MS2 precursor ions were isolated in the quadrupole set to a mass width filter of 1.6 m/z. Ion trap fragmentation spectra (ITMS2) were collected with an AGC target setting of 1.0e4 with a maximum injection time of 35 ms with CID collision energy set at 35%. Raw mass spectrometry data were processed using Proteome Discoverer (v2.2; ThermoFisherScientific) to search against the Uniprot Human Taxonomy database (49,806 entries) with Mascot (v2.6.0; www.matrixscience.com) and Sequest search algorithms. Precursor mass tolerance was set to 20 ppm with fragment mass tolerance set to 0.8 Da with a maximum of two missed cleavages. Modifications: Carbamidomethylation (Cys) was set as fixed and Oxidation (Met) set as variable. Processed LC-MS/MS data files produced database generated files (msf) which were uploaded into Scaffold 4 (v4.10.0) software (www.proteomesoftware.com) to create a.sfd file. The raw data was searched at a stringency threshold of 1% false discovery rate (FDR) for protein and a minimum of one peptide per protein as determined by Mascot and Sequest in the Proteome Discoverer method. The peptide threshold was set to 95%.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD027944 and 10.6019/PXD027944.

Phospho-kinase antibody array: Keratinocytes were treated with integrin inhibitors BOP, BIO1211, LDV, and DMSO as a control for either 0.5 or 6 h before being subjected to Human Phospho-Kinase Antibody Array (R&D Systems, Inc.) as per manufacturer’s instructions.

Western blotting: Western blotting was used for specific protein detection following SDS-PAGE. The proteins were transferred onto PVDF membranes (Sigma-Aldrich) then blocked using 5% BSA/TBST (0.1% Tween (Sigma-Aldrich) in Tris Buffered Saline (TBS)) or 5% milk/TBST for 1 h at room temperature. The membranes were then probed with the primary antibody at 1:1,000 dilution overnight at 4°C. The membranes were then washed prior to incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Proteins were then detected with ECL chemiluminescence kit (Bio-Rad Laboratories) and imaged (ChemiDoc Imaging Systems, Bio-Rad Laboratories). Blots were analyzed and processed using Image Lab (v5.2.1, Bio-Rad Laboratories).

Immunofluorescence and confocal microscopy: Coverslips were coated with Human Plasma Fibronectin Purified Protein (Merck Millipore). Keratinocytes were plated on coverslips either in RM + media, or RM + media without EGF supplement to force colony formation. Once at the correct confluency, cells were treated over variable time points. Cells were then washed and fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS before 10-min permeabilization with 0.2% Triton X-100, followed by blocking with 5% Bovine Serum Albumin (BSA, Roche, Basel, Switzerland) in PBS for 30 min. The cells were labeled with antibodies at 4°C overnight before being stained with secondary antibodies, phalloidin-AlexaFluor, and Hoechst for 1 h at room temperature. The cells were then mounted using Fluorsave Mounting Media (Calbiochem, San Diego, CA, United States) and imaged with a 60x objective using Nikon A1R inverted confocal microscope (Nikon, Minato City, Tokyo, Japan). Images were exported from the Nikon Elements software (Nikon) for further analysis in ImageJ software (United States National Institutes of Health, Bethesda, MD, United States).

Single cell migration assay: Cells were plated in 12 well tissue culture plates containing 1 ml RM + media (1 × 10⁴ cells per well) and allowed to adhere for 6 h at 37°C. Media was then replaced with RM + containing 25 µM HEPES and treatment. Cells were imaged live using IX71 inverted microscope (Olympus, Shinjuku City, Tokyo, Japan) every 15 min for 24 h. TIFF files were imported into ImageJ software (United States National Institutes of Health), where cells were tracked using TrackMate plugin. Data were then imported into Chemotaxis Tool (Ibidi, Gräfelfing, Munich, Germany) for analysis.

Collective cell migration assay: Keratinocytes were plated at confluency and incubated for 2–24 h in the presence of 2 mM of calcium. Confluent monolayers were wounded with a 10 µl pipette tip, and then washed to remove cell debris. Media was then replaced with RM + containing 25 µM HEPES and treatment. Cells were imaged live using IX71 inverted microscope (Olympus) every 15 min for 24 h and were saved as TIFF files. Space between two migration fronts was used as a measure of collective migration. Wound closure was analyzed using ImageJ software (United States National Institutes of Health) a percentage closure at a set time point compared to 0 h. Experiments were performed in the presence of Mitomycin C to control for proliferation changes contributing to migration. No differences were seen between control and Mitomycin C treated cultures in terms of migratory response (not shown).

Proliferation assay: Keratinocytes were plated in RM + media 1 day before the experiment. The cells were then treated with conditioned media containing inhibitors for 24 and 48 h. Cells were then fixed and stained with Hoechst and Phalloidin, before being imaged by tile-scan using EVOSTM FL Auto 2 Imaging System (Thermo Fisher Scientific) with a 10x objective lens. The number of cells in each well was measured by automated nuclei counting using ImageJ software (United States National Institutes of Health).

Colony protrusion assay: Keratinocyte colonies were treated with integrin inhibitors for 24 h to induce protrusion formation. Colonies were then co-treated for 1 h, fixed and stained with Hoechst and Phalloidin. Cells were imaged using EVOS™ FL Auto 2 Imaging System with a 20x objective lens. The percentage increase in area and perimeter of the protrusion from keratinocyte colonies was calculated by subtracting the region enclosed by the cortical actin ring from the binarized total boundaries of the cluster area using ImageJ software (United States National Institutes of Health).

Image analysis: All images were analyzed using ImageJ software and associated plugins (United States National Institutes of Health). To analyze protein signal at the cell periphery or at the leading edge, regions of interest (ROI) were generated to extract the first 5 µm behind the cell boundary. Manders overlap coefficient (MOC) of ROI was calculated using the JACoP plugin. ROIs generated using this method were also used to calculate percentage of internalized α4 integrin in follower cells (cells behind the first two rows adjacent to a scratch); the percentage of internalized α4 integrin = (signal in 5 µm reduced ROI/total signal in ROI around cell periphery) x 100. α9 integrin signal in leader cells was calculated by measuring the basal α9 integrin signal in the first 60 µm behind the leading edge in scratch assays. Nuclear MSK1 signal leader (first 2 rows of cells adjacent to scratch) was calculated by measuring the mean grey value of ROIs generated using the nuclei-stained channel. To count the number of protrusions that extended beyond the leading edge during CMM following inhibitor treatment, protrusion that extended beyond the leading edge during the first 6 h of image acquisition were measured; protrusions that extended more than 20 µm from the leading edge were counted.

Statistical analysis: Data are represented as the mean ± standard error of the mean (s.e.m.). All statistical tests were carried out using Prism package (GraphPad software). The Student’s t-test was performed for comparing two groups for statistical analysis. Analysis of variance (ANOVA) with Sidak post hoc test was used for multiple comparisons. p < 0.05 was considered as statistically significant.

Our study places α4β1 and α9β1 integrins as key regulators of keratinocyte CCM. Efficient wound healing requires a migratory front proximal to the wound to reinstate the barrier, whilst a distal ring of proliferating cells replenishes cells lost following injury (Park et al., 2017). Our data show that inhibition of α4/α9 integrins leads to increased ERK1/2 activation and downstream accumulation of nuclear MSK1. This finding suggests that α4 and α9 integrins may contribute to proliferation zonality during CCM through their localization and signaling; during wounding α4/α9 integrins relocalize to the leading-edge following engagement with components of the provisional ECM (for example, Tenascin-C). This altered distribution activates α4/α9 integrin mediated suppression of ERK1/2 signaling. ERK1/2 suppression could facilitate efficient movement at the migratory front by mediating transient attachment to ECM and cohesively organizing the cytoskeleton (summarized in Figure 5). Conversely, in follower cells, α4 integrins (sequestered at the cell junctions) and α9 integrins (sequestered within the cytoplasm) cannot suppress ERK1/2/MSK signaling, thus promoting normal proliferation rates. Furthermore, previous studies have indicated that α9 integrin regulates proliferation in skin (Singh et al., 2009), gut (Desloges et al., 1998), and lymphatic endothelial cells (Danussi et al., 2013). It will be interesting in future to determine if the pathways we have identified in keratinocytes extend to these other cell types.

Our data further demonstrate that inhibition of α4/α9 integrins increases ERK1/2 activation. This observation agrees with previous studies showing α4/α9 integrin binding to the ligand EMILIN-1 suppresses ERK1/2 signaling (Modica et al., 2017; Danussi et al., 2013; Danussi et al., 2011). Cytoplasmic ERK1/2 can play a role in cell motility by directly phosphorylating and activating several components of the actin cytoskeleton machinery (Tanimura and Takeda 2017), including the WAVE complex (Mendoza et al., 2011) and cortactin (Martinez-Quiles et al., 2004). Furthermore, downstream cytoplasmic ERK1/2 effector RSK can promote motility by activating myosin phosphatase to reduce actin contraction at the leading edge (Samson et al., 2019), and phosphorylating filamin A to inactivate β1 integrins (Gawecka et al., 2012). Furthermore, sustained ERK and MSK signaling induces proliferation (Lavoie, Gagnon, and Therrien 2020). As α4/α9 integrin inhibition results in activation of ERK1/2 signaling, it is likely that dysregulation of ERK1/2, RSK, and MSK may also impact on these pathways resulting in the phenotypes identified in our study.

Next, we sought to address how α4/α9 integrins regulate ERK1/2 signaling. Phosphorylated cytoplasmic ERK must associate with Importin 7 to cross the nuclear envelope (Chuderland, et al 2008). A body of evidence has previously implicated integrins in mediating ERK1/2 nuclear translocation (Hirsch et al., 2002; James et al., 2007) and cell cycle progression (Jeanes et al., 2012). This process offers the possibility that α4/α9 integrins could prevent ERK1/2 translocation by sequestering Importin 7 at the cell membrane. Alternatively, α4/α9 integrins may be regulating ERK activity through trans-dominant action over other integrins. Transdominance is the process by which integrins compete for ECM or cytoplasmic adaptor binding. Integrins with the greatest affinity for either suppress the level of signaling initiated by other integrins that also bind to those adaptors (Gonzalez et al., 2010). α9β1 has previously been shown to trans-dominantly repress α3β1 signaling during wound healing in vitro (DiPersio et al., 2016). Similarly, α4 integrin is trans-dominant over α5 integrins in melanoma cells (Moyano et al., 2003) and in oral squamous carcinoma cells (Zhang et al., 2004). α3β1 and α5β1 are expressed in keratinocytes (Watt and Jones 1993) and are known to induce ERK1/2 phosphorylation (Roovers et al., 1999; Manohar et al., 2004). Hence, α4/α9 integrin inhibition may increase ERK1/2 activation by initiating signaling pathways downstream of these other β1 integrin containing heterodimers. We hypothesize that α4/α9 integrins may therefore locally regulate ERK1/2 activity and nuclear import to prevent proliferation and instead promote migration at the leading edge.

As α4/α9 integrin inhibition suppressed CCM in our study, this inferred that MSK (initially identified in our kinase screen) may have a previously undescribed role in migration. Interestingly, MSK1 inhibition suppressed residual protrusions at the boundary of keratinocyte colonies. Spatiotemporal co-ordination of protrusive activity is essential for migration (Pankov et al., 2005; Dang et al., 2013), and our data suggest that a basal level of MSK signaling is required for optimal cytoskeleton dynamics to permit efficient migration. In support of this interpretation, MSK inhibition and deletion both suppressed CCM. Unexpectedly, MSK1 knockdown also increased protrusions, which suggests that MSK1 may scaffold other protein(s) at the leading edge, which in turn restricts membrane protrusions. This possibility could be supported by the observation that MSK1 was enriched in the protrusive tips of keratinocytes following α4/α9 integrin inhibition. Active MSK1 has principally been studied in the nucleus, and potential cytoplasmic interaction partners or substrates are currently unknown. This topic would be an interesting area for future study and may reveal additional MSK1 dependent targets that play a role in actin dynamics and motility.

In summary, we have identified α4 and α9 integrins as key regulators of CCM in keratinocytes. This study also identifies a previously undescribed role for MSK in the regulation of cytoskeletal dynamics during keratinocyte migration. These findings contribute towards our understanding of the regulation of keratinocyte migration within wound healing, and therefore offer interesting insights into the signaling cascades regulated by this integrin subfamily.