Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility

Srisathya Srinivasan,Sushant Kumar,Ankita Srivastava,Nikita Jain,Chitra Nayak,Rahul Purwar,Sreya Das,Vishakha Surve,Abhijeet Sawant

doi:10.1038/s41598-019-56447-2

Abstract

Actomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

Highlights

Actomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC)
As a first step towards understanding the role of Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) in the migration of healthy and malignant keratinocytes, we examined the effect of inhibition of ROCK and MLCK on the migration of healthy primary keratinocytes (HPKs) and A-431 by using a physiologically relevant 3-dimensional (3D) collagen gel
Y-27632 was used as a ROCK inhibitor, ML-7 as the MLCK inhibitor and Blebbistatin was used for global myosin inhibition

Summary

Introduction

Actomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. Many studies have evaluated the ROCK as a therapeutic target for invasion and metastasis using ROCK specific inhibitors (small molecules), the distinct roles of ROCK and MLCK in regulating NMMIIA activity and their subsequent effects on migration of healthy and malignant human keratinocytes remains unknown. Using specific small molecule inhibitors as well as genetic manipulation techniques, we show that the ROCK pathway is crucial for regulating the contractility, focal adhesion dynamics, actin stress fibre organization and ECM remodelling ability of both HPKs and A-431 cells

Methods

Results

Conclusion