Conditional deficiency of Rho‐associated kinases disrupts endothelial cell junctions and impairs respiratory function in adult mice

• Published in: FEBS open bio 2024-06, Vol.14 (6), p.906-921
• Main Authors: Akamine, Takahiro, Terabayashi, Takeshi, Sasaki, Takako, Hayashi, Riku, Abe, Ichitaro, Hirayama, Fumihiro, Nureki, Shin‐ichi, Ikawa, Masahito, Miyata, Haruhiko, Tokunaga, Akinori, Kobayashi, Takashi, Hanada, Katsuhiro, Thumkeo, Dean, Narumiya, Shuh, Ishizaki, Toshimasa
• Format: Article
• Language: English
• Subjects: Actin; Actin Cytoskeleton; Alveoli; Animals; Blood pressure; Catenin; Cell activation; Cell Adhesion; Cell junctions; Cell migration; cell–cell adhesion; Cytoskeleton; Embryogenesis; Endothelial cells; Heart rate; Homeostasis; Homology; Kinases; Laboratories; lung function; Lungs; Permeability; Phalloidin; Phosphorylation; Polymerase chain reaction; Proteins; Respiratory function; ROCK; Transcription activation; transgenic mice; Tumor necrosis factor-TNF; vascular permeability; β-Catenin
• ISSN: 2211-5463; 2211-5463
• DOI: 10.1002/2211-5463.13802

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho‐associated coiled‐coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)‐inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc‐CreERT2) were generated and administered a 5‐day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double‐floxed control mice. On Day 3 post‐TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post‐TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)‐cadherin, β‐catenin, and p120‐catenin at cell–cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE‐cadherin/β‐catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell–cell adhesion. Tamoxifen‐induced Rho‐associated coiled‐coil kinase 1 and 2 double knockout (ROCK1/2 DcKO) in adult mice caused the disruption of endothelial barrier in their lungs. In addition, severe pulmonary hemorrhagic plaques were observed. As results, respiratory function was impaired. These findings indicated that ROCK proteins play a crucial role in maintaining lung function by regulating endothelial barrier in vivo.