TRPV4 regulates matrix stiffness and TGFβ1‐induced epithelial‐mesenchymal transition

Abstract Substrate stiffness (or rigidity) of the extracellular matrix has important functions in numerous pathophysiological processes including fibrosis. Emerging data support a role for both a mechanical signal, for example, matrix stiffness, and a biochemical signal, for example, transforming growth factor β1 (TGFβ1), in epithelial‐mesenchymal transition (EMT), a process critically involved in fibrosis. Here, we report evidence showing that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel, is the likely mediator of EMT in response to both TGFβ1 and matrix stiffness. Specifically, we found that: (a) genetic ablation or pharmacological inhibition of TRPV4 blocked matrix stiffness and TGFβ1‐induced EMT in normal mouse primary epidermal keratinocytes (NMEKs) as determined by changes in morphology, adhesion, migration and alterations of expression of EMT markers including E‐cadherin, N‐cadherin (NCAD) and α‐smooth muscle actin (α‐SMA), and (b) TRPV4 deficiency prevented matrix stiffness‐induced EMT in NMEKs over a pathophysiological range. Intriguingly, TRPV4 deletion in mice suppressed expression of mesenchymal markers, NCAD and α‐SMA, in a bleomycin‐induced murine skin fibrosis model. Mechanistically, we found that: (a) TRPV4 was essential for the nuclear translocation of YAP/TAZ (yes‐associated protein/transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1, (b) TRPV4 deletion inhibited both matrix stiffness‐ and TGFβ1‐induced expression of YAP/TAZ proteins and (c) TRPV4 deletion abrogated both matrix stiffness‐ and TGFβ1‐induced activation of AKT, but not Smad2/3, suggesting a mechanism by which TRPV4 activity regulates EMT in NMEKs. Altogether, these data identify a novel role for TRPV4 in regulating EMT.

(EMT) is a cell differentiation process by which polarized epithelial cells undergo biochemical/molecular changes, losing their cell polarity and cell-cell adhesion and gaining migratory capacity, invasiveness and elevated resistance to apoptosis, thereby assuming a mesenchymal phenotype. [9][10][11][12][13] EMT has essential functions in fundamental cellular processes including embryogenesis, development, tissue repair and oncogenesis. [9][10][11][12][13] However, unchecked and exacerbated EMT can contribute to various pathological conditions such as metastasis and tissue fibrosis. [9][10][11][12][13] Fibrotic diseases including pulmonary, liver and skin fibrosis are characterized by an increase in the invasion and migration of mesenchymal cells across a stiffened ECM associated with induction of EMT. [12][13][14] Transforming growth factor β1 (TGFβ1)-induced EMT in primary human keratinocytes and altered expression of various EMT markers in skin tissues from scleroderma, keloids and melanoma patients have recently been demonstrated, indicating a link between EMT and skin fibrosis and oncogenesis. [15][16][17][18][19] Mechanical cues, for example, stiffness, in the ECM can influence cellular functions such as cell spreading, motility, differentiation, proliferation, and apoptosis, which are also regulated by TGFβ1. 7,[20][21][22][23] Emerging data support a role for matrix stiffness in EMT in epithelial cells. 16,[21][22][23] Zarkoob et al 16 have demonstrated specific effect of matrix stiffness on keratinocyte colony formation. Furthermore, it has been reported that calcium signalling and expression of specific Ca 2+ -permeable ion channels are involved in induction of EMT. [24][25][26][27][28] However, role of matrix stiffness and/or calcium signalling-induced EMT in skin keratinocytes has not been reported. Although the signalling mechanisms underlying EMT have been well studied, the identity of a matrix stiffness-sensing plasma membrane receptor/channel and the molecular mechanisms by which matrix stiffness signals are transmitted and propagated to drive EMT remain to be determined. Recently, we reported that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive Ca 2+ -permeable channel, is associated with skin and lung fibrosis.
TRPV4 regulates both biochemical (TGFβ1)-and mechanical (matrix stiffness) stimulus-induced lung and dermal myofibroblast differentiation and contributes to the development of in vivo pulmonary and skin fibrosis in murine models. [29][30][31] However, the specific role of TRPV4 in EMT in normal primary epidermal keratinocytes has not been determined.

| TRPV4 mediates TGFβ1-induced EMT-like changes in normal mouse primary epidermal keratinocytes
To determine whether TRPV4 channels are required in EMT, we first determined the presence of functional Ca 2+ permeable TRPV4 channels in normal mouse primary epidermal keratinocytes (NMEKs) by comparing Ca 2+ influx in NMEKs derived from TRPV4 KO to those of wild-type (WT) mice. We recorded a concentrationdependent (1-1000 nmol/L) increase in Ca 2+ influx in WT NMEKs by a selective TRPV4 agonist, GSK101 (EC 50 = 5 nmol/L), which was not seen in TRPV4 KO NMEKs ( Figure 1A-C). 29,61 These results confirmed the presence of functional TRPV4 calcium-permeable channels in NMEKs, as reported previously. 38,39 Immunofluorescent and immunoblot staining indicated the presence of TRPV4 proteins in NMEKs ( Figure 1D,E). Together, these results indicate that functional Ca 2+ permeable TRPV4 channels are expressed in NMEKs.
Transforming growth factor β1 is known to induce EMT in keratinocytes. 15 Recent evidence from our group suggests a link between TRPV4 activation and TGFβ1 signals that mediate fibroblast differentiation. [29][30][31] Previous reports have documented that calcium signalling is involved in EMT in many cell types. [24][25][26][27][28] However, the identity of a plasma membrane calcium channel and the mechanism by which calcium signals are transduced/propagated in cells to drive EMT, are not well understood. To test our hypothesis that TRPV4 activation might regulate EMT, NMEKs from WT and TRPV4 KO mice were treated with TGFβ1, and compared for acquisition of EMT-like phenotypic properties or EMT-related biochemical changes. We found that compared to untreated WT controls, TGFβ1 pre-treated WT NMEKs showed down-regulated ECAD expression along with up-regulated α-SMA expression (  It has been reported that increases in matrix stiffness drive TGFβ1mediated EMT in mammary gland epithelial cells. 23 However, the identity of a matrix stiffness sensing calcium channel and the mechanism by which calcium signals are transduced/propagated into cells to drive EMT are not known. To ascertain the role of TRPV4 on EMT in response to increasing matrix stiffness alone or in combination with TGFβ1, we seeded NMEKs and HDFs on compliant (0.5 kPa) or stiff (8 and 50 kPa) polyacrylamide hydrogels treated with or without TGFβ1, and assessed the occurrence of EMT-like changes in TRPV4 functiondeficient and WT groups. We found that TGFβ1 was unable to drive morphological changes in WT NMEKs under compliant conditions (normal skin tissue stiffness) ( Figure 3A

| TRPV4 deletion blocks EMT marker expression in a mouse model of bleomycin-induced skin fibrosis
We previously reported that TRPV4 KO mice are protected from pro-fibrotic effects of bleomycin in lung and skin, 29,30 and bleomycin has been shown to cause EMT in skin and pulmonary fibrosis model. 17,62 To determine whether TRPV4 deficiency could also suppress EMT in an experimental model of skin fibrosis, we employed  Recent evidence suggests that matrix stiffness and TGFβ1 cooperate to induce renal fibrogenesis in a YAP/TAZ-dependent manner. 63 Previously, it was reported that YAP and TAZ pre-dominantly localize to the nucleus to drive EMT under high stiffness. 58 However, the identity of a matrix stiffness sensing mechanotransduction receptor/channel and the mechanism by which mechanosensing signals are transduced/propagated into cells to drive YAP/TAZ nuclear transduction and subsequently EMT are not known. As expected, we found that stiff matrix (8 kPa) alone induced an increase in the nuclear localization of YAP and TAZ in WT NMEKs ( Figure 6A,B).
Pre-treatment with TGFβ1 further amplified the stiffness-induced YAP/TAZ nuclear accumulation in WT NMEKs compared to WT NMEKs without TGFβ1 treatment ( Figure 6A,B). The absence of TRPV4 reduced the localization of YAP/TAZ to the nucleus in NMEKs grown on stiff substrate with or without TGFβ1 ( Figure 6A, B). These findings suggest that TRPV4 regulates YAP and TAZ activity in response to both matrix stiffness and TGFβ1. We assessed translocation of TAZ to nucleus by immunofluorescence in WT NMEKs pre-treated with blebbistatin ( Figure 6C,D). These results indicate that blebbistatin pre-treatment reduced TAZ nuclear translocation, suggesting TRPV4 may promote TAZ nuclear localization via cytoskeletal remodelling.
We further investigated the effect of matrix stiffness and TGFβ1 on the expression levels of YAP and TAZ by immunoblot analysis using two different antibodies that recognize YAP and TAZ separately. In WT NMEKs, treatment with TGFβ1 pre-dominantly increased the expression level of TAZ compared to untreated WT controls ( Figure 7A,B). The expression levels of YAP in TGFβ1-treated WT NMEKs remained unchanged compared to untreated WT controls ( Figure 7A,B). However, the absence of TRPV4 significantly decreased YAP and TAZ expression levels when compared to WT NMEKs with or without TGFβ1 ( Figure 7A,B). There was no effect of TGFβ1 on YAP or TAZ expression levels between untreated and TGFβ1-treated TRPV4 KO NMEKs ( Figure 7A,B). Similarly, stiff matrix (25 kPa) caused a significant increase in TAZ expression levels in WT NMEKs, which was completely absent in TRPV4 KO NMEKs ( Figure 7C,D). We also determined the impact of matrix stiffness on phosphorylation of Lats1, a Hippo pathway kinase. Immunoblot analysis show positive effects of stiffness (1 vs. 25 kPa) on phosphorylation of Lats1 in WT NMEKs; however, this effect was not dependent on TRPV4 ( Figure 7E). Altogether, these findings suggest that TRPV4 is an essential regulator of YAP and TAZ expression, the

| Deletion of TRPV4 channel blocks AKT activation required for EMT
Activation of the PI3K/AKT pathway is a central promoter of EMT in many cell types. 64,65 The PI3K/AKT pathway can be activated in either a TGFβ1-dependent or -independent manner. [49][50][51] It has been shown that activation of the PI3K/AKT pathway is up-regulated by stiff matrices. 23,66 We found that TGFβ1-induced activation of Smad-2/3 proteins in a time-dependent manner in both WT and TRPV4 KO NMEKs ( Figure 8A,B). Furthermore, we found that endogenous expression and phosphorylation of Smad2/3 was increased in TRPV4 KO NMEKs treated or not with TGFβ1 compared to WT NMEKs.
These results suggest that TRPV4 may act as a negative regulator of endogenous expression and phosphorylation of Smad2/3. However, genetic deletion of TRPV4 significantly suppressed phosphorylation of AKT (at serine 473) with or without TGFβ1 compared to WT NMEKs ( Figure 8A,B), suggesting that both basal and TGFβ1-induced activation of AKT are dependent on TRPV4. We compared phosphorylated AKT (p-AKT) levels between WT and TRPV4 KO NMEKs grown on soft (1 kPa) and stiff (25 kPa) matrices. We found that stiff matrices caused increased p-AKT levels in WT NMEKs compared to WT NMEKs grown on soft matrix ( Figure 8C,D). In contrast, we found a complete absence of p-AKT in TRPV4 KO NMEKs grown on either soft or stiff matrices ( Figure 8C,D). These findings suggest that the absence of TRPV4 in NMEKs abrogates both stiffness and TGFβ1-induced phosphorylation of AKT.

| DISCUSSION
The key findings described herein are: (a) TRPV4, a mechanosensitive Data are expressed as mean ± SEM, n = 5 mice/group; ns = non-significant; ***P < 0.001, one-way ANOVA. (C) Representative immunofluorescence staining of PBS and Bleo skin sections from WT mice for TRPV4 (green) and EMT markers (ECAD and NCAD; red). Nuclei were stained with DAPI (blue). In merge, yellow colour indicates co-localization of TRPV4 and NCAD. (D) Quantitation of results from (C). Data are expressed as mean ± SEM, n = 5 mice/group; **P < 0.01, ***P < 0.001, one-way ANOVA markers in a murine skin fibrosis model and (c) fibrosis induced in skin tissues with bleomycin is associated with increased co-localization of TRPV4 with mesenchymal markers and decreased co-localization of TRPV4 with epithelial markers. We found that: (a) TRPV4 is essential for the nuclear translocation of YAP and TAZ in response to matrix stiffness and TGFβ1 in NMEKs; (b) TRPV4 deletion blocks both matrix stiffness-induced and TGFβ1-induced expression of YAP and TAZ proteins and (c) TRPV4 deletion abrogates matrix stiffness-induced and TGFβ1-induced activation of AKT, but not Smad2/3, suggesting a mechanism by which TRPV4 activity regulates EMT. These findings identify a novel role for TRPV4 in regulating EMT.
Here, we found that TRPV4 is essential for the nuclear translocation of YAP/TAZ in response to matrix stiffness and TGFβ1 in NMEKs, suggesting a possible regulatory role of TRPV4 mechanosensing in YAP/TAZ transcriptional activity. The increased expression of YAP and TAZ proteins may modulate nuclear availability of these proteins under multiple pathophysiological conditions. 52-60 Here, we found that TRPV4 deletion blocked both matrix stiffness-induced and TGFβ1-induced expression of YAP and TAZ proteins in NMEKs, suggesting an additional mechanism of regulating YAP/TAZ activity by TRPV4. It has been reported that matrix stiffness regulates YAP/TAZ localization to the nucleus by increasing cytoskeletal tension. 57,58 Previously, we reported that TGFβ1-induced cytoskeletal remodelling in fibroblasts is regulated in part by TRPV4. 29 Here, we found that TAZ nuclear localization in response to TGFβ1 is dependent on cytoskeletal remodelling. Our current results suggest that TRPV4 may promote EMT in NMEKs via cytoskeletal remodelling that regulates YAP/TAZ nuclear translocation.
We found that both WT and TRPV4 KO NMEKs were sensitive to TGFβ1-induced Smad2/3 activation. This suggests that TRPV4 mediates EMT via a Smad-independent pathway. However, both stiff matrices and TGFβ1-induced activation of AKT in WT NMEKs, which was absent in TRPV4 KO NMEKs. It is known that TGFβ1-induced EMT is regulated by activation of the PI3K/AKT pathway. [49][50][51]71,73 It is also reported that increasing stiffness regulates TGFβ1-induced EMT through PI3K/AKT signalling. 23 17,62 Our present data showed that TRPV4 is associated with bleomycin-induced EMT. This is in agreement with our recent finding that TRPV4 deletion protects mice against bleomycin-induced skin fibrosis. 30 Intracellular calcium plays an important role in every aspect of cellular life including inducing EMT. [24][25][26][27][28]74,75 Calcium signalling and the expression of specific Ca 2+ -permeable ion channels are involved in induction of proteins associated with EMT. [24][25][26][27][28] The identification of the role of specific Ca 2+ -permeable ion channels in the induction of EMT in the context of fibrosis and oncogenesis suggests that these channels may be appropriate therapeutic targets for control of EMT-mediated disease progression. Our recently published reports suggested that TRPV4-dependent Ca 2+ influx potentiates TGFβ1induced lung and dermal myofibroblast differentiation in response to matrix stiffness, and showed that TRPV4 KO mice were protected from bleomycin-induced fibrosis in skin and lungs. [29][30][31] Although TGFβ1 and matrix stiffness were shown to play important roles in EMT and fibrosis, the role of TRPV4 mechanosensing in EMT has not been reported. Our data are consistent with a model ( Figure 8E) in which TRPV4-dependent Ca 2+ influx integrates matrix stiffness and soluble signals to promote EMT via YAP/TAZ and PI3K/AKT pathways, and thus may contribute to development of fibrosis and oncogenesis. However, our results warrant further studies to determine whether AKT activation is downstream to YAP/TAZ pathway. It has been reported that matrix stiffness regulates YAP/TAZ translocation to nucleus by increasing cytoskeletal tension, and the activation of Hippo pathway and the subcellular distribution of YAP is also influenced by cell attachment status. 76 In our model we found that matrix stiffness-induced phosphorylation of Lats1 is not dependent on TRPV4, suggesting this channel has no direct role in regulating Lats1 activation.
It is also unknown how TRPV4 is sensitized by matrix stiffness or TGFβ1. In view of the fact that TRPV4 mediates actin polymerization and integrin signalling, 29,40,41 it will be interesting to determine if TRPV4 is sensitized through cytoskeletal remodelling and/or integrin signalling via a feed-forward mechanism. In summary, we report a novel role of TRPV4 channels in regulating matrix stiffness-induced and TGFβ1-induced EMT in normal skin epithelial cells. Furthermore understanding the mechanism by which TRPV4 regulates EMT will support therapeutic targeting of TRPV4 signalling to combat fibrosis, foreign body response and oncogenesis.

| Cell culture
Primary normal mouse epidermal keratinocytes were derived from the tail of 8-10-week-old WT and TRPV4 KO adult mice as published previously. 79 Briefly, the mouse tail skin was washed with sterile cold PBS, and treated with trypsin (0.25%) for 2 hours at 37°C, 5% CO 2 to separate epidermis and dermis. The peeled epidermis was rinsed thoroughly in PBS and minced in cold keratinocyte specific media (ATCC, Manassas, VA, USA). The cell suspension was pipetted up and down several times, strained, centrifuged and resuspended in media. NMEKs were then seeded in collagen-coated (10 μg/ml) culture flasks. Medium was replaced every 48 hours thereafter. NMEKs were maintained in complete keratinocyte media supplemented with keratinocyte growth kit (ATCC) and penicillin/ streptomycin (Thermo Fisher Scientific). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 and sub-cultured at 70% confluence. Purity of NMEKs was examined by changes in morphology and by analysing alterations of expression of epithelial/keratinocyte specific markers as previously published. 9 For all experiments, NMEKs were seeded on collagen coated (10 μg/ml) plastic or polyacrylamide hydrogels with compliant (0.5 and 1 kPa) and stiff (8 and 25 kPa) matrices followed by treatment with vehicle or TGFβ1 (5 ng/ml).

| Bleomycin-induced murine skin fibrosis model
The bleomycin-induced skin fibrosis model was employed as described previously. 29,30 Bleomycin was prepared by dissolving bleomycin sulfate (Hospira, Lake Forest, IL, USA) in sterile PBS.
Briefly, 6-month-old WT and TRPV4 KO mice (n = 5 per group) received equal volumes (0.1 ml) of bleomycin (10 mg/kg) or PBS (control). All injections were administered subcutaneously to the shaved dorsal area of the mice every alternate day for 28 days. All mice were euthanized 24 hours after the last dose, and skin tissues were harvested for immunofluorescence staining. Skin samples were snap frozen in liquid nitrogen, embedded in OCT (Sakura Finetek, USA), and stored at −80°C. Cryostat sections (7 μm) were mounted on glass slides.

| Intracellular calcium influx assay
Changes in intracellular calcium (Ca 2+ ) or calcium influx in NMEKs was performed on the FlexStation3 system using a FLIPR calcium 5 Assay Kit as previously described. 29 Briefly, NMEKs (15 000 cells/ well in complete keratinocyte media) were treated in 96-well plates with or without TGFβ1 at 37°C, 5% CO 2 . After 24 hours, cells were incubated with FLIPR kit reagents (calcium 5 dye in 1X HBSS solution containing 20 mmol/L HEPES and 2.5 mmol/L probenecid) for 45 minutes at 37°C, followed by incubation with vehicle or TRPV4 antagonist GSK2193874 (GSK219) for 45 minutes at 37°C. 80 Plates were incubated in the dark for another 10 minutes and then transferred to the FlexStation3 system to measure fluorescence. Calcium influx was induced by the TRPV4 agonist GSK1016790A (GSK101) 61 in vehicle-pretreated or GSK219-pretreated NMEKs, and cytosolic Ca 2+ influx was recorded by measuring ΔF/F (Max-Min) as described previously. 29,31 Data are shown as relative fluorescence units.

| Scratch wound healing assay and cell spreading
For scratch wound healing assay, wounded HDF monolayers were incubated in 1% BSA containing serum-free medium with or without the indicated antagonists. Images of the wounds were captured at 0.5 hours (time 0) and 24 or 48 hours later, and the total number of migrated cells in the "wound" areas was counted. To assess morphological changes in WT, TRPV4 KO NMEKs and TRPV4 antagonisttreated MDFs, cells were seeded on collagen-coated polyacrylamide hydrogels with compliant (0.5 and 1 kPa) or stiff (8,25, and 50 kPa) matrices. Cells were incubated with TGFβ1, antagonists or vehicle in complete keratinocyte or MDF media, for 72 hours. Cells were examined by phase contrast or fluorescence microscopy (Carl Zeiss, Germany) for phenotypic changes related to EMT. Images were captured, and percent EMT, cell adherence and spread area were calculated.

| Immunofluorescence staining
Normal mouse primary epidermal keratinocytes were grown on collagen coated cover glass or polyacrylamide hydrogels, and treated with or without TGFβ1 for 72 hours. Cells were fixed with 3% SHARMA ET AL. | 771 paraformaldehyde, permeabilized with 0.1% Triton X-100, washed and blocked with 3% BSA/PBS. Cells were immunostained for ECAD, α-SMA, TRPV4, YAP and TAZ. Bleomycin or vehicle treated mouse skin sections were fixed with acetone, and immunostained for α-SMA, NCAD, ECAD and TRPV4, followed by incubation with Alexa Fluor 488 or 594 conjugated IgG. Cells were then mounted using Prolong diamond antifade reagent with DAPI. Immunofluorescence intensity was quantified using ImageJ software (NIH), and the results were expressed as integrated density (int. density) (the product of area and mean grey value).

| Statistical analysis
All data are expressed as mean ± SEM. Statistical analysis was performed with the Student's t test for two groups or ANOVA for three or more groups using Prism software. A value of P ≤ 0.05 was considered statistically significant.