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Cellular and Molecular Bioengineering

, Volume 12, Issue 2, pp 139–152 | Cite as

The TRPV4-TAZ Mechanotransduction Signaling Axis in Matrix Stiffness- and TGFβ1-Induced Epithelial-Mesenchymal Transition

  • Shweta Sharma
  • Rishov Goswami
  • Shaik O. RahamanEmail author
Article

Abstract

Introduction

The implantation of biomaterials into soft tissue leads to the development of foreign body response, a non-specific inflammatory condition that is characterized by the presence of fibrotic tissue. Epithelial–mesenchymal transition (EMT) is a key event in development, fibrosis, and oncogenesis. Emerging data support a role for both a mechanical signal and a biochemical signal in EMT. We hypothesized that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel, is a mediator of EMT.

Methods

Normal human primary epidermal keratinocytes (NHEKs) were seeded on collagen-coated plastic plates or varied stiffness polyacrylamide gels in the presence or absence of TGFβ1. Immunofluorescence, immunoblot, and polymerase chain reaction analysis were performed to determine expression level of EMT markers and signaling proteins. Knock-down of TRPV4 function was achieved by siRNA transfection or by GSK2193874 treatment.

Results

We found that knock-down of TRPV4 blocked both matrix stiffness- and TGFβ1-induced EMT in NHEKs. In a murine skin fibrosis model, TRPV4 deletion resulted in decreased expression of the mesenchymal marker, α-SMA, and increased expression of epithelial marker, E-cadherin. Mechanistically, our data showed that: (i) TRPV4 was essential for the nuclear translocation of TAZ in response to matrix stiffness and TGFβ1; (ii) Antagonism of TRPV4 inhibited both matrix stiffness-induced and TGFβ1-induced expression of TAZ proteins; and (iii) TRPV4 antagonism suppressed both matrix stiffness-induced and TGFβ1-induced activation of Smad2/3, but not of AKT.

Conclusions

These data identify a novel role for TRPV4-TAZ mechanotransduction signaling axis in regulating EMT in NHEKs in response to both matrix stiffness and TGFβ1.

Keywords

TRPV4 Epithelial–mesenchymal transition Keratinocytes Matrix stiffness TAZ Smad2/3 Fibrosis 

Notes

ACKNOWLEDGMENTS

Startup grant from University of Maryland, NIH (1R01EB024556-01), and NSF (CMMI-1662776) grants to Shaik O. Rahaman.

AUTHOR CONTRIBUTIONS

SS and SOR conceived the study, designed and performed the experiments, and wrote the manuscript. RG assisted with experiments and analysis of data, and maintained the animal colony. All authors reviewed the results and approved the final content of the manuscript.

Conflict of interest

Shweta Sharma, Rishov Goswami, and Shaik O. Rahaman declare that they have no conflicts of interest.

ETHICAL APPROVAL

The study protocol was approved by the University of Maryland Review Committee, and all experiments were performed in accordance with the IACUC guidelines.

INFORMED CONSENT

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

12195_2018_565_MOESM1_ESM.pdf (3 mb)
Supplementary Fig. 1 TRPV4 antagonism suppresses Ca2+ influx and modulates expression of matrix stiffness and TGFβ1-induced ECAD and α-SMA. (A) FlexStation 3 recording of Calcium 6 dye-loaded NHEK monolayers assessing effects of TRPV4 selective antagonist, GSK219, on Ca2+ influx induced by calcium ionophore, A23 (2 μM) or TRPV4 selective agonist, GSK101 (20 nM). Bar graph is showing the quantified results (mean ± SEM). All experiments were performed 3 times in quadruplicate. **p < 0.01; 1-way ANOVA. (B and C) NHEKs were plated on collagen-coated (10 μg/mL) plastic plates and were incubated with or without GSK219 for 24 h. qRT-PCR analysis was performed to determine ECAD, GAPDH, and Vimentin mRNA levels using SYBR Green gene Expression Assay. Ct values were normalized to GAPDH levels. **p < 0.01; 1-way ANOVA. (D) NHEKs were plated on soft (1 kPa) or stiff (25 kPa) polyacrylamide hydrogels coated with collagen (10 μg/mL), and were incubated with or without TGFβ1 (5 ng/mL) for 96 h. For this experiment, we refreshed the media with GSK219 (5 nM) every 24 h. The data shown is one of the representative images from four different fields per condition to assess the capacity of TRPV4 inhibition (by GSK219) to inhibit matrix stiffness and TGFβ1-induced increases in the expression of ECAD and α-SMA. ECAD (red), α-SMA (green), and DAPI (blue) stains are shown. Scale bars: 10 µm. (E) Quantitation of results shown in D. Data are expressed as mean ± SEM of three independent experiments, n = 20 cells/condition. ns = non-significant; **p < 0.01, ***p < 0.001; 1-way ANOVA. hpf: high power field. (PDF 3096 kb)
12195_2018_565_MOESM2_ESM.pdf (6.6 mb)
Supplementary Fig. 2 TRPV4 inhibition by siRNA modulates expression of matrix stiffness and TGFβ1-induced ECAD and α-SMA. (A) NHEKs were plated on soft (1 kPa) or stiff (25 kPa) collagen-coated (10 μg/mL) polyacrylamide hydrogels, and were transfected with scrambled siRNA (Scr) or TRPV4 specific siRNA (si-TRPV4) for 96 h. ECAD (red), α-SMA (green), and DAPI (blue) stains are shown. Scale bars: 10 µm. (B) Quantitation of results shown in A. Data are expressed as mean ± SEM of three independent experiments, n = 20 cells/condition. ns = non-significant; *p < 0.05, **p < 0.01, ***p < 0.001; 1-way ANOVA. hpf: high power field. (PDF 6799 kb)

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Copyright information

© Biomedical Engineering Society 2018

Authors and Affiliations

  • Shweta Sharma
    • 1
  • Rishov Goswami
    • 1
  • Shaik O. Rahaman
    • 1
    Email author
  1. 1.Department of Nutrition and Food ScienceUniversity of MarylandCollege ParkUSA

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