ROCK1 Mediates Retinal Glial Cell Migration Promoted by Acrolein

Abstract

Objective: Acrolein is a highly reactive aldehyde that covalently binds to cellular macromolecules and subsequently modulates cellular function. Our previous study demonstrated that acrolein induces glial cell migration, a pathological hallmark of diabetic retinopathy; however, the detailed cellular mechanism remains unclear. The purpose of this study was to investigate the role of acrolein in retinal glial cell migration by focusing on rho-associated coiled-coil-containing protein kinases (ROCKs).Methods: Immunofluorescence staining for ROCK isoforms was performed using sections of fibrovascular tissue obtained from the eyes of patients with proliferative diabetic retinopathy (PDR). Rat retinal Müller glial cell line, TR-MUL5, was stimulated with acrolein and the levels of ROCK1 were evaluated using real-time PCR and western blotting. Phosphorylation of the myosin-binding subunit of myosin light chain phosphatase [myosin phosphatase target subunit 1, (MYPT1)] and myosin light chain 2 (MLC2) was assessed. The cell migration rate of TR-MUL5 cells exposed to acrolein and/or ripasudil, a non-selective ROCK inhibitor, was measured using the Oris cell migration assay.Results: ROCK isoforms, ROCK1 and ROCK2, were positively stained in the cytosol of glial cells in fibrovascular tissues. In TR-MUL5 cells, the mRNA expression level of Rock1, but not Rock2, was increased following acrolein stimulation. In line with the PCR data, western blotting showed increase in ROCK1 and cleaved ROCK1 protein in TR-MUL5 cells stimulated with acrolein. N-acetylcysteine (NAC) suppressed acrolein-associated Rock1 upregulation in TR-MUL5 cells. Acrolein augmented the phosphorylation of MYPT1 and MLC2 and increased the cell migration rate of TR-MUL5 cells, both of which were abrogated by ripasudil.Conclusions: Our study demonstrated that ROCK1 mediates the migration of retinal glial cells promoted by the unsaturated aldehyde acrolein.

Acrolein, an endogenous aldehyde induces synaptic dysfunction in vitro and in vivo: Involvement of RhoA/ROCK2 pathway

“…ROCK1 is mainly distributed in peripheral tissues such as the lung, liver, and spleen, and ROCK2 is mainly distributed in the heart and nervous system (Yan et al, 2019 ). Recently, a study reported that ROCK1 mediates the migration of glial cells promoted by acrolein (Fukutsu et al, 2021 ). Although RNA‐Seq results and our previous acrolein‐conjucated proteomics data indicated that ROCK pathway might be involved in acrolein toxicity.…”

“…Recent studies indicated that ROCK2 is a promising therapeutic target for AD (Cai et al, 2021 ; Weber & Herskowitz, 2021 ). Some studies indicated that acrolein is closely related to ROCK1 (Fukutsu et al, 2021 ) but no relevant biological experiment conducted to substantiate the relationship between acrolein‐induced early AD and ROCK2 in the brain. In this study, acrolein could activate the RhoA/ROCK2/p‐cofilin pathway both in vivo and vitro (Figure 4 ).…”

Diet as a Source of Acrolein: Molecular Basis of Aldehyde Biological Activity in Diabetes and Digestive System Diseases

“…Of course, acrolein-dependent oxidative stress has further molecular targets. Other factors that are not without significance in diabetes and the accompanying DN, DR, and neurodegeneration include acrolein-dependent chronic inflammation, damage to blood vessels, the dysfunction of Müller cells, and the effect on cell division and proliferation [ 80 , 81 , 82 , 83 ].…”

“…Valuable information on acrolein-dependent glial cell migration and the pathological hallmark of diabetic retinopathy is provided by the latest research from the research team of Fukutsu et al [ 80 , 118 ]. In their research on rat retinal Müller glial cell line TR-MUL5, they focused on the key retinal glial cell migration and activation of rho-associated coiled-coil-containing protein kinases (ROCKs) [ 80 , 118 ]. Activation of the ROCK-1 signaling pathway induces, among others, focal vascular constrictions, endoluminal blebbing, and subsequent retinal hypoxia [ 119 ].…”

“…As emphasized in these studies, a significant increase in the expression of the ROCK1 isoform and activation of the kinase following acrolein stimulation was observed. The consequence of increased ROCK1 activity was that acrolein dependence enhances phosphorylation of myosin phosphatase target subunit 1 (MYPT1) and myosin light-chain 2 (MLC2) through the ROCK1 cascade induced by oxidative stress [ 80 , 118 ].…”