Rho-associated Protein Kinase Pathway Research Articles

Mechanistic study for drug induced cholestasis using batch-fabricated 3D spheroids developed by agarose-stamping method

Cell spheroid culture can recapitulate the tissue microstructure and cellular responses in vivo. While there is a strong need to understand the modes of toxic action using the spheroid culture method, existing preparation techniques suffer from low efficiency and high cost. Herein, we developed a metal stamp containing hundreds of protrusions for batch bulk preparation of cell spheroids in each well of the culture plates. The agarose matrix imprinted by the stamp can form an array of hemispherical pits, which facilitated the fabrication of hundreds of uniformly sized rat hepatocyte spheroids in each well. Chlorpromazine (CPZ) was used as a model drug to investigate the mechanism for drug induced cholestasis (DIC) by agarose-stamping method. Hepatocyte spheroids showed a more sensitive detection of hepatotoxicity compared to 2D and Matrigel-based culture systems. Cell spheroids were also collected for staining of cholestatic protein and showed a CPZ-concentration-dependent decrease of bile acid efflux related proteins (BSEP and MRP2) and tight junction (ZO-1). In addition, the stamping system successfully delineated the DIC mechanism by CPZ that may be associated with the phosphorylation of MYPT1 and MLC2, two central proteins in the Rho-associated protein kinase pathway (ROCK), which were significantly attenuated by ROCK inhibitors. Our results demonstrated a large-scale fabrication of cell spheroids by the agarose-stamping method, with promising benefits for exploring the mechanisms for drug hepatotoxic responses.

Vanillic Acid Alleviates Right Ventricular Function in Rats With MCT-Induced Pulmonary Arterial Hypertension

This study examined the molecular processes behind the effects of vanillic acid (VA) on right ventricular (RV) hypertrophy and function in rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). There were 40 male Sprague‒Dawley (SD) rats that were separated into 4 groups: Control, PAH, MCT + VA (50 mg/kg/d), and MCT + VA (100 mg/kg/d). Male SD rats were injected with MCT once under the skin to create the PAH model (40 mg/kg). RV morphological properties were evaluated using Masson and hematoxylin and eosin (H&E) staining. Echocardiography was used to evaluate RV functioning and right ventricle–pulmonary artery (RV-PA) coupling. In addition, Rho-associated protein kinase (ROCK) pathway-related factors were evaluated using Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to detect inflammatory markers as well as atrial natriuretic peptide (ANP) and brain-type natriuretic peptide (BNP) in the blood of PAH rats. As a result, VA effectively reduced the development of RV cardiomyocyte hypertrophy and fibrosis in PAH rats; levels of ANP, BNP, and inflammatory markers in the blood of PAH rats were also significantly decreased by VA intervention. Additionally, VA enhanced RV functioning and RV-PA coupling in PAH rats. In response to VA, the expression of proteins related to the ROCK pathway (ROCK1, ROCK2, NFATc3, P-STAT3, and Bax) was downregulated, whereas Bcl-2 expression was elevated. This study found that VA could attenuate RV remodeling and improve RV-PA coupling in PAH rats. RV remodeling and dysfunction may be linked to the dysregulation of the ROCK pathway, and the protective action of VA on RV function may be due to a block in the ROCK signaling pathway or its downstream signaling molecules.

Tacrolimus Causes Hypertension by Increasing Vascular Contractility via RhoA (Ras Homolog Family Member A)/ROCK (Rho-Associated Protein Kinase) Pathway in Mice.

To provide tacrolimus is first-line treatment after liver and kidney transplantation. However, hypertension and nephrotoxicity are common tacrolimus side effects that limit its use. Although tacrolimus-related hypertension is well known, the underlying mechanisms are not. Here, we test whether tacrolimus-induced hypertension involves the RhoA (Ras homolog family member A)/ROCK (Rho-associated protein kinase) pathway in male C57Bl/6 mice. Intra-arterial blood pressure was measured under anesthesia. The reactivity of renal afferent arterioles and mesenteric arteries were assessed in vitro using microperfusion and wire myography, respectively. Tacrolimus induced a transient rise in systolic arterial pressure that was blocked by the RhoA/ROCK inhibitor Fasudil (12.0±0.9 versus 3.2±0.7; P<0.001). Moreover, tacrolimus reduced the glomerular filtration rate, which was also prevented by Fasudil (187±20 versus 281±8.5; P<0.001). Interestingly, tacrolimus enhanced the sensitivity of afferent arterioles and mesenteric arteries to Ang II (angiotensin II), likely due to increased intracellular Ca2+ mobilization and sensitization. Fasudil prevented increased Ang II-sensitivity and blocked Ca2+ mobilization and sensitization. Preincubation of mouse aortic vascular smooth muscle cells with tacrolimus activated the RhoA/ROCK/MYPT-1 (myosin phosphatase targeting subunit 1) pathway. Further, tacrolimus increased cytoplasmic reactive oxygen species generation in afferent arterioles (107±5.9 versus 163±6.4; P<0.001) and in cultured mouse aortic vascular smooth muscle cells (100±7.5 versus 160±23.2; P<0.01). Finally, the reactive oxygen species scavenger Tempol inhibited tacrolimus-induced Ang II hypersensitivity in afferent arterioles and mesenteric arteries. The RhoA/ROCK pathway may play an important role in tacrolimus-induced hypertension by enhancing Ang II-specific vasoconstriction, and reactive oxygen species may participate in this process by activating the RhoA/ROCK pathway.

Reducing Proteoglycan Synthesis and NOX Activity by ROCK Inhibitors: Therapeutic Targets in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arteries characterized by the accumulation of inflammatory cells in the arterial wall. Hypertension, dyslipidemia, and hyperglycemia are major risk factors of atherosclerosis. Rho-associated protein kinase (ROCK), a serine/threonine kinase, is a downstream effector of the small GTPase RhoA. ROCK is involved in different stages of atherosclerosis. Accumulating evidence has demonstrated that ROCK signaling plays vital roles in various cellular functions, such as contraction, migration, and proliferation of smooth muscle cells. Dysregulation of the ROCK pathway is associated with atherosclerosis and hypertension. Experimental studies have shown that ROCK inhibitors may have favorable effects in ameliorating atherosclerosis. ROCK signaling has a role in proteoglycan synthesis through transactivation of the TGF-β receptor Type I (TβRI) mediated by G-protein-coupled receptor (GPCR) agonists (endothelin-1, angiotensin II and …), and ROCK inhibitors could decrease proteoglycan synthesis and atherosclerotic plaque formation. Based on the hypothesis that targeting ROCK pathway may be effective in ameliorating atherosclerosis, we suggest that ROCK inhibitors may have a potential therapeutic role in inhibition or slowing atherogenesis. However, for this hypothesis more research is needed.

Cadmium exposure enhances VE-cadherin expression in endothelial cells via suppression of ROCK signaling.

Vascular endothelium is a target of cadmium (Cd), which is a global pollutant of the environment. However, the detailed effects and underlying mechanisms remain to be elucidated. In the present study, human umbilical vein endothelial cells (HUVECs) were treated with 0.1, 1, 5, 10, 50 µM cadmium chloride (CdCl2) for 12 h. It was found that vascular endothelial (VE)-cadherin mRNA and protein expression was upregulated by Cd in HUVECs in a dose-dependent manner. Higher levels of VE-cadherin were detected at cell-to-cell junctions in HUVECs treated with 10 µM CdCl2 compared with normal condition. The phosphorylation level of myosin-binding subunit of myosin phosphatase, a downstream substrate of Rho-associated protein kinase (ROCK), was reduced by 10 µM CdCl2, suggesting that Cd inhibited the Rho/ROCK pathway. Activation of ROCK by narciclasine reversed the Cd-induced increase of VE-cadherin expression. By contrast, ROCK pathway inhibitor Y27632 increased VE-cadherin expression in HUVECs. Following inhibition of the ROCK pathway, Cd did not significantly alter the level of VE-cadherin. Taken together, the results suggested that Cd exposure enhanced VE-cadherin expression in endothelial cells via suppression of ROCK signaling.

Role of Rho-kinase Inhibitor and hrsACE2 in COVID-19 Patients Management

The Renin-angiotensin system (RAS) plays an important role in the SARS-CoV-2 infection through Angiotensin-converting enzyme 2 (ACE2). Human recombinant soluble ACE2 (hrsACE2) has been shown to act as a competitive interceptor for SARS-CoV-2 by preventing the binding of viral particles to the ACE2. In addition, Rho-kinase inhibitors have been proven to suppress SARS-CoV-2 infection through inhibition of the Rho-associated protein kinase (ROCK) pathway. The combined effect of hrsACE2 and Rho-kinase inhibitor increases the activity and levels of ACE2 in protection through the Ang1-7 MasR, Ang1-9, and other mechanisms of protection. This literature review is done by searching journals with “COVID-19”, “ACE2”, “hrsACE2”, “Rho- kinase inhibitor”, and “ROCK pathway” as well as Boolean logic “AND” and “OR”. Relevant journals are used as references to compile systematic writing. Based on research results, hrsACE2 and Rho-kinase inhibitors have been clinically proven to protect tissue through the elevated level of ACE2 by the mechanism of Rho-kinase inhibitor, which increased Ang1-7 MasR concentration that gives vasodilatation, anti-proliferative, anti-inflammation, and anti-fibrotic effect. Rho-kinase inhibitors significantly reduce the number of infected cells by SARS-CoV-2 in COVID-19 patients. The potential of the combination therapy of Rho-kinase inhibitor and hrsACE2 therapy can be an efficient therapeutic solution for COVID-19 patients so that further research can be carried out in the future.

Inflammation and Rho-Associated Protein Kinase-Induced Brain Changes in Vascular Dementia.

Patients with vascular dementia, caused by cerebral ischemia, experience long-term cognitive impairment due to the lack of effective treatment. The mechanisms of and treatments for vascular dementia have been investigated in various animal models; however, the insufficient information on gene expression changes that define pathological conditions hampers progress. To investigate the underlying mechanism of and facilitate treatment development for vascular dementia, we established a mouse model of chronic cerebral hypoperfusion, including bilateral carotid artery stenosis, by using microcoils, and elucidated the molecular pathway underlying vascular dementia development. Rho-associated protein kinase (ROCK) 1/2, which regulates cellular structure, and inflammatory cytokines (IL-1 and IL-6) were upregulated in the vascular dementia model. However, expression of claudin-5, which maintains the blood–brain barrier, and MAP2 as a nerve cell-specific factor, was decreased in the hippocampal region of the vascular dementia model. Thus, we revealed that ROCK pathway activation loosens the tight junction of the blood–brain barrier and increases the influx of inflammatory cytokines into the hippocampal region, leading to neuronal death and causing cognitive and emotional dysfunction. Our vascular dementia model allows effective study of the vascular dementia mechanism. Moreover, the ROCK pathway may be a target for vascular dementia treatment development in the future.

Synaptotoxicity in Alzheimer's Disease Involved a Dysregulation of Actin Cytoskeleton Dynamics through Cofilin 1 Phosphorylation.

Amyloid-β (Aβ) drives the synaptic impairment and dendritic spine loss characteristic of Alzheimer's disease (AD), but how Aβ affects the actin cytoskeleton remains unknown and contentious. The actin-binding protein, cofilin-1 (cof1), is a major regulator of actin dynamics in dendritic spines, and is subject to phospho-regulation by multiple pathways, including the Rho-associated protein kinase (ROCK) pathway. While cof1 is implicated as a driver of the synaptotoxicity characteristic of the early phases of AD pathophysiology, questions remain about the molecular mechanisms involved. Cofilin-actin rods are observed in neurons exposed to Aβ oligomers (Aβo) and in tissue from AD patients, and others have described an increased cofilin phosphorylation (p-cof1) in AD patients. Here, we report elevated p-cof1 of the postsynaptic enriched fraction of synaptosomes from cortical samples of male APP/PS1 mice and human AD cases of either sex. In primary cortical neurons, Aβo induced rapid actin stabilization and increased p-cof1 in the postsynaptic compartment of excitatory synapses within 30 min. Fluorescence recovery after photobleaching of actin-GFP and calcium imaging in live neurons expressing active or inactive cof1 mutants suggest that cof1 phosphorylation is necessary and sufficient for Aβo-induced synaptic impairment via actin stabilization before the reported formation of cofilin-actin rods. Moreover, the clinically available and well-tolerated ROCK inhibitor, fasudil, prevented Aβo-induced actin stabilization, synaptic impairment, and synaptic loss by blocking cofilin phosphorylation. Aβo also blocked the LTP-induced insertion of the AMPAR subunit, GluA1, at the postsynaptic density, in a fasudil-sensitive manner. These data support an important role for ROCKs and cofilin in mediating Aβ-induced synaptic impairment.SIGNIFICANCE STATEMENT We report that amyloid-β oligomers rapidly induce aberrant stabilization of F-actin within dendritic spines, which impairs synaptic strength and plasticity. Activation of the Rho-associated protein kinase (ROCK) pathway results in phosphorylation of cof1 and is sufficient to mediate Aβo-induced actin stabilization synaptic impairment and synaptic loss. Further, the ROCK inhibitor, fasudil, prevents cofilin phosphorylation, acute synaptic disruption, and synaptotoxicity in primary cortical neurons. Together, the herein presented data provide strong support for further study of the ROCK pathway as a therapeutic target for the cognitive decline and synaptotoxicity in Alzheimer's disease.

Blockade of Rho-associated protein kinase (ROCK) inhibits the contractility and invasion potential of cancer stem like cells.

Recent studies have implicated the roles of cancer stem like cells (CSCs) in cancer metastasis. However, very limited knowledge exists at the molecular and cellular level to target CSCs for prevention of cancer metastasis. In this study, we examined the roles of contractile dynamics of CSCs in cell invasion and delineated the underlying molecular mechanisms of their distinct cell invasion potential. Using de-adhesion assay and atomic force microscopy, we show that CSCs derived from melanoma and breast cancer cell lines exhibit increased contractility compared to non-CSCs across all tumor types. In addition, CSCs possess increased ECM remodeling capacity as quantified by collagen degradation assay. More importantly, pharmacological blockade of Rho-associated protein kinase completely abolished the contractility and collagen degradation capacity of both CSCs and non-CSCs. In conclusion, our study demonstrates the importance of cell contractility in regulating invasiveness of CSCs and suggests that pharmacological targeting of ROCK pathway represents a novel strategy for targeting both CSCs and bulk population for the treatment of cancer metastasis.

Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells.

Lines of evidence suggest that Rho-associated protein kinase (ROCK)-mediated myosin phosphatase-targeting subunit 1 (MYPT1) phosphorylation plays a central role in smooth muscle contraction. However, the physiological significance of MYPT1 phosphorylation at Thr696 catalyzed by ROCK in bladder smooth muscle remains controversial. We attempt to directly observe the quantitative protein expression of Rho A/ROCK and phosphorylation of MYPT1 at Thr696 after carbachol administration in rat bladder smooth muscle cells (RBMSCs). Primary cultured smooth muscle cells were obtained from rat bladders. The effects of both concentration and time-course induced by the muscarinic agonist carbachol were investigated by assessing the expression of Rho A/ROCK and MYPT1 phosphorylation at Thr696 using Western blot. In the dose-course studies, carbachol showed significant increase in phosphorylation of MYPT1 at Thr696 (p-MYPT1) from concentrations of 15-100μM based on Western blot results (p<0.05, ANOVA test). In the time-course studies, treatment of cells with 15μM of carbachol significantly enhanced the expression of p-MYPT1 from 3 to 15h (p<0.05, ANOVA test) and induced the expression of Rho A from 10 to 120min (p<0.05, ANOVA test). Carbachol can induce the expression of ROCK pathway, leading to MYPT1 phosphorylation at Thr696 and thereby sustained RBSMCs contraction.

Down-regulation of cell adhesion via rho-associated protein kinase (ROCK) pathway promotes tumor cell migration on laminin-511

Epithelial cells, both normal and precancerous, stably anchor to basement membranes, whereas malignant tumors pass through them to achieve metastasis. Of basement membrane components, laminin-511 (α5, β1, γ1; LM-511) has been found to be a major isoform in many adult basement membranes. Several studies have shown that LM-511 promotes not only cell adhesion but also tumor cell migration. Thus, LM-511 can be viewed like two distinct molecules in normal vs. tumor cells; tumor cells seem to be able to alter their response (adhesive vs. migratory) to LM-511. In this study we examined the effects of biologically active molecules on A549 lung adenocarcinoma cell adhesion to LM-511. Of them, phorbol 12-myristate 13-acetate (PMA) induced transition to a rounded cell shape and significantly promoted cell migration on LM-511. The attachment of PMA-treated A549 cells to LM-511 was weaker than that of control cells. PMA-stimulated signaling pathway reduced the binding of integrin α3β1 to LM-511. Cell migration assays using inhibitors for signal transduction and cytoskeletal organization showed that suppression of cell adhesion via the rho-associated protein kinase (ROCK) pathway promoted tumor cell migration on LM-511. Our results suggest that the ROCK pathway is involved in the transition from static to migratory cell behaviors on LM-511.

RhoA orchestrates glycolysis for TH2 cell differentiation and allergic airway inflammation

Mitochondrial metabolism is known to be important for T-cell activation. However, its involvement in effector T-cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T-cell activation and effector cell differentiation and function remains largely unknown. We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for TH2 cell differentiation and TH2-mediated allergic airway inflammation. Conditional RhoA-deficient mice were generated by crossing RhoA(flox/flox) mice with CD2-Cre transgenic mice. Effects of RhoA on TH2 differentiation were evaluated based on invitro TH2-polarized culture conditions and invivo in ovalbumin-induced allergic airway inflammation. Cytokine levels were measured by using intracellular staining and ELISA. T-cell metabolism was measured by using the Seahorse XF24 Analyzer and flow cytometry. Disruption of RhoA inhibited T-cell activation and TH2 differentiation invitro and prevented the development of allergic airway inflammation invivo, with no effect on TH1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways, such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to TH2 cell differentiation and allergic airway inflammation through regulating IL-4 receptor mRNA expression and TH2-specific signaling events. Finally, inhibition of Rho-associated protein kinase, an immediate downstream effector of RhoA, blocked TH2 differentiation and allergic airway inflammation. RhoA is a key component of the signaling cascades leading to TH2 differentiation and allergic airway inflammation at least in part through control of T-cell metabolism and the Rho-associated protein kinase pathway.

Forgetting to remember in epilepsy

Amyloid-β (Aβ) drives the synaptic impairment and dendritic spine loss characteristic of Alzheimer9s disease (AD), but how Aβ affects the actin cytoskeleton remains unknown and contentious. The actin-binding protein, cofilin-1 (cof1), is a major regulator of actin dynamics in dendritic spines, and is subject to phospho-regulation by multiple pathways, including the Rho-associated protein kinase (ROCK) pathway. While cof1 is implicated as a driver of the synaptotoxicity characteristic of the early phases of AD pathophysiology, questions remain about the molecular mechanisms involved. Cofilin-actin rods are observed in neurons exposed to Aβ oligomers (Aβo) and in tissue from AD patients, and others have described an increased cofilin phosphorylation (p-cof1) in AD patients. Here, we report elevated p-cof1 of the postsynaptic enriched fraction of synaptosomes from cortical samples of male APP/PS1 mice and human AD cases of either sex. In primary cortical neurons, Aβo induced rapid actin stabilization and increased p-cof1 in the postsynaptic compartment of excitatory synapses within 30 min. Fluorescence recovery after photobleaching of actin-GFP and calcium imaging in live neurons expressing active or inactive cof1 mutants suggest that cof1 phosphorylation is necessary and sufficient for Aβo-induced synaptic impairment via actin stabilization before the reported formation of cofilin-actin rods. Moreover, the clinically available and well-tolerated ROCK inhibitor, fasudil, prevented Aβo-induced actin stabilization, synaptic impairment, and synaptic loss by blocking cofilin phosphorylation. Aβo also blocked the LTP-induced insertion of the AMPAR subunit, GluA1, at the postsynaptic density, in a fasudil-sensitive manner. These data support an important role for ROCKs and cofilin in mediating Aβ-induced synaptic impairment. <b>SIGNIFICANCE STATEMENT</b> We report that amyloid-β oligomers rapidly induce aberrant stabilization of F-actin within dendritic spines, which impairs synaptic strength and plasticity. Activation of the Rho-associated protein kinase (ROCK) pathway results in phosphorylation of cof1 and is sufficient to mediate Aβo-induced actin stabilization synaptic impairment and synaptic loss. Further, the ROCK inhibitor, fasudil, prevents cofilin phosphorylation, acute synaptic disruption, and synaptotoxicity in primary cortical neurons. Together, the herein presented data provide strong support for further study of the ROCK pathway as a therapeutic target for the cognitive decline and synaptotoxicity in Alzheimer9s disease.