Identification of a small RhoA GTPase inhibitor effective in fission yeast and human cells.

The Rho GTPase family proteins are key regulators of cytoskeletal dynamics. Deregulated activity of Rho GTPases is associated with cancers and neurodegenerative diseases, and their potential as drug targets has long been recognized. Using an economically effective drug screening workflow in fission yeast and human cells, we have identified a Rho GTPase inhibitor, O1. By a suppressor mutant screen in fission yeast, we find a point mutation in the rho1 gene that confers resistance to O1. Consistent with the idea that O1 is the direct inhibitor of Rho1, O1 reduced the cellular amount of activated, GTP-bound Rho1 in wild-type cells, but not in the O1-resistant mutant cells, in which the evolutionarily conserved Ala62 residue is mutated to Thr. Similarly, O1 inhibits activity of the human orthologue RhoA GTPase in tissue culture cells. Our studies illustrate the power of yeast phenotypic screens in the identification and characterization of drugs relevant to human cells and have identified a novel GTPase inhibitor for fission yeast and human cells.

Fucoxanthin decreases lipopolysaccharide-induced acute lung injury through the inhibition of RhoA activation and the NF-κB pathway.

Fucoxanthin is a natural pigment widely distributed in macroalgae and microalgae. An orange-colored xanthophyll, it has several bioactive effects, including anticancer, anti-obesity, oxidative stress reduction, and anti-inflammation. Acute lung injury (ALI) caused by acute infections or injurious stimuli to the lung tissues is a severe pulmonary inflammatory disease. To date, no evidence has shown ALI to be reduced by fucoxanthin through activation of Ras homolog family member A (RhoA) and the nuclear factor (NF)-κB pathway in lipopolysaccharide (LPS)-treated mice. Pretreatment with fucoxanthin inhibited histopathological changes in lung tissues and neutrophil infiltration into bronchoalveolar lavage fluid induced by LPS in ALI mice. Moreover, LPS-induced proinflammatory cytokine expression and neutrophil infiltration were inhibited by fucoxanthin in a concentration-dependent manner. Pretreatment of mice with fucoxanthin inhibited NF-κB phosphorylation and IκB degradation in the lungs of mice with LPS-induced ALI. We further found that phosphorylation of Akt and p38 mitogen-activated protein KINASE (MAPK) was inhibited by fucoxanthin. By contrast, the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase was not inhibited by fucoxanthin. Furthermore, we found that the activation of RhoA was inhibited by fucoxanthin in LPS-induced ALI. On the basis of these results, we propose that fucoxanthin disrupts the RhoA activation-mediated phosphorylation of Akt and p38 MAPK, leading to NF-κB activation in mice with LPS-induced ALI.

FPR2 participates in epithelial ovarian cancer (EOC) progression through RhoA-mediated M2 macrophage polarization.

BACKGROUND: In our previous study, we found that formyl peptide receptor 2 (FPR2) promoted the invasion and metastasis of epithelial ovarian cancer (EOC) and could be a prognostic marker for EOC. In this study, we aimed to study the possible mechanism of FPR2 in promoting EOC progression. METHODS: EOC cell lines with ectopic FPR2 expression and knockdown as well as their control cell lines were established, and the expression change of RhoA in each cell line was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) and Western blot. Wound healing and Transwell assays were performed to detect the migratory ability of EOCs affected by FPR2 and RhoA. The supernatant of each EOC cell line was used to coculture with macrophages, and then we tested M1 and M2 macrophage biomarkers in the supernatants by flow cytometry. The THP-1 cell line was also induced to differentiate into M1 and M2 macrophages, and FPR2 and RhoA expression in each macrophage cell line was detected by RT-qPCR and Western blot. A tumour xenograft model was established with SKOV3 and SKOV3-shFPR2 cell lines, and tumour volumes and weights were recorded. RESULTS: RhoA expression was significantly increased in EOCs along with the overexpression of FPR2, which showed a positive correlation by Pearson correlation analysis. Ectopic FPR2 expression contributes to the migratory ability of EOCs, and a RhoA inhibitor (C3 transferase) impairs EOC migration. Furthermore, FPR2 stimulated the secretion of Th2 cytokines by EOCs, which induced macrophages to differentiate to the M2 phenotype, while a RhoA inhibitor stimulated the secretion of Th1 cytokines and induced macrophages to differentiate to the M1 phenotype. Moreover, compared with M1 macrophages and THP-1 cells, FPR2 and RhoA expression was significantly upregulated in M2 macrophages. CONCLUSION: FPR2 stimulated M2 macrophage polarization and promoted invasion and metastasis of ovarian cancer cells through RhoA.

SH3-Binding Glutamic Acid Rich-Deficiency Augments Apoptosis in Neonatal Rat Cardiomyocytes.

Congenital heart disease (CHD) is one of the most common birth defects in humans, present in around 40% of newborns with Down's syndrome (DS). The SH3 domain-binding glutamic acid-rich (SH3BGR) gene, which maps to the DS region, belongs to a gene family encoding a cluster of small thioredoxin-like proteins sharing SH3 domains. Although its expression is confined to the cardiac and skeletal muscle, the physiological role of SH3BGR in the heart is poorly understood. Interestingly, we observed a significant upregulation of SH3BGR in failing hearts of mice and human patients with hypertrophic cardiomyopathy. Along these lines, the overexpression of SH3BGR exhibited a significant increase in the expression of hypertrophic markers (Nppa and Nppb) and increased cell surface area in neonatal rat ventricular cardiomyocytes (NRVCMs), whereas its knockdown attenuated cellular hypertrophy. Mechanistically, using serum response factor (SRF) response element-driven luciferase assays in the presence or the absence of RhoA or its inhibitor, we found that the pro-hypertrophic effects of SH3BGR are mediated via the RhoA-SRF axis. Furthermore, SH3BGR knockdown resulted in the induction of apoptosis and reduced cell viability in NRVCMs via apoptotic Hippo-YAP signaling. Taking these results together, we here show that SH3BGR is vital for maintaining cytoskeletal integrity and cellular viability in NRVCMs through its modulation of the SRF/YAP signaling pathways.

Disrupting RhoA activity by blocking Arhgef3 expression mitigates microglia-induced neuroinflammation post spinal cord contusion.

Excess inflammatory microglia activation deteriorates the pathological degree of spinal cord injury (SCI). We here employed microglia samples in vitro and murine model in vivo to trace the role of inhibition of Arhgef3 in inflammatory response post SCI. From the specimen analysis of lipopolysaccharide (LPS)-induced inflammatory microglia, we found that Arhgef3 expression was positively relative to microglia activation. In vitro, LPS caused the microglia inflammatory activation and induced upregulation of the Arhgef3 expression. Interestingly, presence of Arhgef3 could activate RhoA through promoting Rho GTPases, but silencing of Arhgef3 decreased RhoA activation and inhibited the microglia inflammation. Moreover, disruption of Arhgef3 inhibited the GTP-RhoA, resulted in a suppression of proinflammatory cytokines, and alleviated the LPS-elicited inflammatory genes expression. Moreover, artificially decreasing Arhgef3 expression remarkedly reduced ROS generation after LPS treatment. In vivo of a mouse mechanical contusion-induced SCI model, inhibition of Arhgef3 reduced the ratio of GTP-RhoA/Total-RhoA, and prevented SCI via mitigating the microglial inflammatory phenotype and decreased secondary neurological injury. Besides, inhibition of Arhgef3 prevented alleviated the degree of demyelination but did not affect neuronal regeneration. Meaningfully, absence of Arhgef3 improved mouse locomotor recovery post SCI. Taken together, Arhgef3 involves the microglial activation and inflammatory response following neural injury, and targeted disrupting of which may indicate a promising therapeutic direction in preventing SCI.

Emerging targets in drug discovery against neurodegenerative diseases: Control of synapsis disfunction by the RhoA/ROCK pathway.

Synaptic spine morphology is controlled by the activity of Rac1, Cdc42 and RhoA, which need to be finely balanced, and in particular RhoA/ROCK prevents the formation of new protrusions by stabilizing actin formation. These processes are crucial to the maturation process, slowing the de novo generation of new spines. The RhoA/ROCK also influences plasticity processes, and selective modulation by ROCK1 of MLC-dependent actin dynamics leads to neurite retraction, but not to spine retraction. ROCK1 is also responsible for the reduction of the readily releasable pool of synaptic vesicles. These and other evidences suggest that ROCK1 is the main isoform acting on the presynaptic neuron. On the other hand, ROCK2 seems to have broad effects on LIMK/cofilin-dependent plasticity processes such as cofilin-dependent PSD changes. The RhoA/ROCK pathway is an important factor in several different brain-related pathologies via both downstream and upstream pathways. In the aggregate, these evidences show that the RhoA/ROCK pathway has a central role in the etiopathogenesis of a large group of CNS diseases, which underscores the importance of the pharmacological modulation of RhoA/ROCK as an important pathway to drug discovery in the neurodegenerative disease area. This article aims at providing the first review of the role of compounds acting on the RhoA/ROCK pathway in the control of synaptic disfunction.

Strategies to neutralize RhoA/ROCK pathway after spinal cord injury.

Regeneration is bungled following CNS injuries, including spinal cord injury (SCI). Inherent decay of permissive conditions restricts the regrowth of the mature CNS after an injury. Hypertrophic scarring, insignificant intrinsic axon-growth activity, and axon-growth inhibitory molecules such as myelin inhibitors and scar inhibitors constitute a significant hindrance to spinal cord repair. Besides these molecules, a combined absence of various mechanisms responsible for axonal regeneration is the main reason behind the dereliction of the adult CNS to regenerate. The neutralization of specific inhibitors/proteins by stymieing antibodies or encouraging enzymatic degradation results in improved axon regeneration. Previous efforts to induce regeneration after SCI have stimulated axonal development in or near lesion sites, but not beyond them. Several pathways are responsible for the axonal growth obstruction after a CNS injury, including SCI. Herein, we summarize the axonal, glial, and intrinsic factor which impedes the regeneration. We have also discussed the methods to stabilize microtubules and through this to maintain the proper cytoskeletal dynamics of growth cone as disorganized microtubules lead to the failure of axonal regeneration. Moreover, we primarily focus on diverse inhibitors of axonal growth and molecular approaches to counteract them and their downstream intracellular signaling through the RhoA/ROCK pathway.

16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro.

Reciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition.

Dexmedetomidine alleviates cardiomyocyte apoptosis and cardiac dysfunction may be associated with inhibition of RhoA/ROCK pathway in mice with myocardial infarction.

The global incidence of myocardial infarction has been consistently high, and it is one of the main causes of poor cardiovascular prognosis. Dexmedetomidine (DEX) is a highly selective α2 receptor agonist. Recent studies have found that DEX has a protective effect on myocardial infarction, but its specific mechanism is still unclear. In this experiment, we permanently ligated the anterior descending branch of mice to explore the protective mechanism of DEX against myocardial infarction. Our study found that intraperitoneal injection of DEX for 7 days after myocardial infarction in mice can increase the reduction of ejection fraction (EF) and fractional shortening (FS) caused by myocardial infarction and significantly reduce the release of serum markers. The results of myocardial HE and Sirius red staining suggest that the changes in the myocardial structure of mice after using DEX are reduced. Immunohistochemistry shows that DEX reduces the expression of ROCK1 protein after myocardial infarction. TUNEL staining and the protein expression levels of cleaved caspase-3 and cleaved caspase-9 were used to detect cell apoptosis and results make clear that DEX can reduce the apoptosis caused by myocardial infarction. Western blot experiments showed that DEX can reduce the expression levels of ROCK1 and ROCK2 (Rho-kinase). At the same time, it was observed that DEX improved the Bcl-2/Bax ratio. The above results indicate that DEX reduces cardiomyocyte apoptosis and improves cardiac function likely through inhibiting the RhoA/ROCK signaling pathway. This study may provide new insights into the protective effect of DEX after myocardial infarction in mice.

The distinct roles of myosin IIA and IIB under compression stress in nucleus pulposus cells.

OBJECTIVES: Inappropriate or excessive compression applied to intervertebral disc (IVD) contributes substantially to IVD degeneration. The actomyosin system plays a leading role in responding to mechanical stimuli. In the present study, we investigated the roles of myosin II isoforms in the compression stress-induced senescence of nucleus pulposus (NP) cells. MATERIAL AND METHODS: Nucleus pulposus cells were exposed to 1.0 MPa compression for 0, 12, 24 or 36 hours. Immunofluorescence and co-immunoprecipitation analysis were used to measure the interaction of myosin IIA and IIB with actin. Western blot analysis and immunofluorescence staining were used to detect nuclear expression and nuclear localization of MRTF-A. In addition, the expression levels of p-RhoA/RhoA, ROCK1/2 and p-MLC/MLC were measured in human NP cells under compression stress and in degenerative IVD tissues. RESULTS: Compression stress increased the interaction of myosin IIA and actin, while the interaction of myosin IIB and actin was reduced. The actomyosin cytoskeleton remodelling was involved in the compression stress-induced fibrotic phenotype mediated by MRTF-A nuclear translocation and inhibition of proliferation in NP cells. Furthermore, RhoA/ROCK1 pathway activation mediated compression stress-induced human NP cells senescence by regulating the interaction of myosin IIA and IIB with actin. CONCLUSIONS: We for the first time investigated the regulation of actomyosin cytoskeleton in human NP cells under compression stress. It provided new insights into the development of therapy for effectively inhibiting IVD degeneration.

Exploring the potential of RhoA inhibitors to improve exercise-recoverable spinal cord injury: A systematic review and meta-analysis.

BACKGROUND: The spinal cord is one of the central nervous system. Spinal cord injury (SCI) will cause loss of physical function and dysfunction below the injury site, causing them to lose sensation and mobility, thereby reducing the quality of life of patients. Although regular rehabilitation management can reduce its severity, the current effective treatment methods are limited to the treatment of secondary injuries to SCI. The purpose of treatment should not only include the restoration of the histology of the lesion, but also should focus on the restoration of sensory and mobility and. The key to effective treatment is to reduce secondary injuries. RhoA inhibitor can improve the pathophysiological changes related to secondary injury and promote the recovery of activity ability, so it may become a clinical drug for the treatment of SCI. This article systematically analyzed the effects of RhoA inhibitors on the promotion of axon regeneration and the recovery of mobility and compared the therapeutic effects of different inhibitors on SCI and their effects on physical function recovery. METHODS: We used a meta-analysis to systematically evaluate the effects of Rho inhibitors on SCI treatment and the recovery of body function. RESULTS: 21 articles (738 animals) were identified in the literatures search. Studies were selected if they reported the therapeutic effects of RhoA/ROCK inhibitors (BA-210, EGCG, ß-elemene, C3-exoenzmye, LINGO-1-Fc, Ibuprofen, SiRhoA, iRhoA + FK506, Fasudil, p21Cip1/WAF1, HA-1007, Y-27,632 and C3bot154-182). We measure the functional recovery by BBB and BMS scores. The random effect model of weighted mean difference (WMD, 95 % confidence interval) was used to analyze the effects. The WMD of the forest graph was 2.277; 95 % CI: 1.705∼2.849, P < 0.001, suggesting that RhoA inhibitors can effectively treat SCI. In addition to EGCG, all the other agents also showed the effects on the activity recovery post-SCI (P < 0.05). CONCLUSION: ß-elemene, LINGO-1-Fc, Ibuprofen, SiRhoA, RhoA + FK506, Fasudil, p21Cip1/WAF1 and Y-27,632 have similar effects to BA-210, they can promote axon germination and nerve fiber regeneration after thoracic spinal cord injury and reduce the formation of syringomyelia and protect white matter, thereby improving locomotor recovery. RhoA inhibitors have great potential to restore motor function and provide a new trend for the treatment of SCI.

Role of RhoA-ROCK signaling in Parkinson's disease.

Parkinson's disease (PD) is a complex and widespread neurodegenerative disease characterized by depletion of midbrain dopaminergic (DA) neurons. Key issues are the development of therapies that can stop or reverse the disease progression, identification of dependable biomarkers, and better understanding of the pathophysiological mechanisms of PD. RhoA-ROCK signals appear to have an important role in PD symptoms, making it a possible approach for PD treatment strategies. Activation of RhoA-ROCK (Rho-associated coiled-coil containing protein kinase) appears to stimulate various PD risk factors including aggregation of alpha-synuclein (αSyn), dysregulation of autophagy, and activation of apoptosis. This manuscript reviews current updates about the biology and function of the RhoA-ROCK pathway and discusses the possible role of this signaling pathway in causing the pathogenesis of PD. We conclude that inhibition of the RhoA-ROCK signaling pathway may have high translational potential and could be a promising therapeutic target in PD.

A high expression ratio of RhoA/RhoB is associated with the migratory and invasive properties of basal-like Breast Tumors.

Basal-like breast cancer is among the most aggressive cancers and there is still no effective targeted treatment. In order to identify new therapeutic targets, we performed mRNA-Seq on eight breast cancer cell lines. Among the genes overexpressed in basal-like tumors, we focused on the RhoA and RhoB genes, which encode small GTPases known to play a role in the actin cytoskeleton, allowing cells to migrate. qRT-PCR and Western blotting were used for expression studies. Migratory and invasive properties were analysed by wound healing and Boyden chambers assays. Stress fibers formation was evaluated by fluorescent actin labeling. Rho siRNA, small inhibitor Rhosin treatment and BRCA1 transfection were performed to study the role of Rho and BRCA1 proteins. We showed that strong expression of RhoA and low expression of RhoB was associated with the basal-like subtype of breast cancer. Decreasing RhoA expression reduced the migratory and invasive capacities of basal-like cell lines, while decreasing RhoB expression increased these capacities. Rhosin, an inhibitor of RhoA, could also reduce the migration of basal-like cell lines. Rho proteins are involved in the formation of stress fibers, a conformation of the actin cytoskeleton found in migrating cells: inhibition of RhoA expression decreased the formation of these fibers. BRCA1, a gene frequently inactivated in basal-like tumors, appears to play a role in the differential expression of RhoA and RhoB in these tumors, as the restoration of BRCA1 expression in a BRCA1-mutated basal-like cell line decreased expression of RhoA and increased expression of RhoB, resulting in reduced migratory capacity. These results suggest Rho proteins as potential therapeutic targets for basal-like and BRCA1-mutated breast cancer, as migration and acquisition of mesenchymal properties are key functional pathways in these tumors with high metastatic potential.

Small molecule modulation of the p75 neurotrophin receptor inhibits multiple amyloid beta-induced tau pathologies.

Longitudinal preclinical and clinical studies suggest that Aß drives neurite and synapse degeneration through an array of tau-dependent and independent mechanisms. The intracellular signaling networks regulated by the p75 neurotrophin receptor (p75NTR) substantially overlap with those linked to Aß and to tau. Here we examine the hypothesis that modulation of p75NTR will suppress the generation of multiple potentially pathogenic tau species and related signaling to protect dendritic spines and processes from Aß-induced injury. In neurons exposed to oligomeric Aß in vitro and APP mutant mouse models, modulation of p75NTR signaling using the small-molecule LM11A-31 was found to inhibit Aß-associated degeneration of neurites and spines; and tau phosphorylation, cleavage, oligomerization and missorting. In line with these effects on tau, LM11A-31 inhibited excess activation of Fyn kinase and its targets, tau and NMDA-NR2B, and decreased Rho kinase signaling changes and downstream aberrant cofilin phosphorylation. In vitro studies with pseudohyperphosphorylated tau and constitutively active RhoA revealed that LM11A-31 likely acts principally upstream of tau phosphorylation, and has effects preventing spine loss both up and downstream of RhoA activation. These findings support the hypothesis that modulation of p75NTR signaling inhibits a broad spectrum of Aß-triggered, tau-related molecular pathology thereby contributing to synaptic resilience.

RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization.

BACKGROUND: In this study, we investigated the mechanism of Rho GTPases signaling on Ang II-mediated cell migration and dedifferentiation in human aortic vascular smooth muscle cells (HA-VSMCs) and an Ang II-infusion mouse model. METHODS: Cells were pretreated with different inhibitors or Ang II. Cell migration was detected by Wound healing and Transwell assay. Mice were treated with Ad-RhoA-shRNA virus or Irbesartan or fasudil and then infused with Ang II. RESULTS: Ang II treatment induced HA-VSMCs migration in a dose- and time-dependent manner and reduced the expression of VSMC contractile proteins. These effects were significantly suppressed by the inhibition of Ang II type 1 receptor (AT1 receptor), RhoA, and Rho-associated kinase (ROCK). Furthermore, Ang II treatment promoted the activation of RhoA and ROCK, which was reduced by AT1 receptor inhibition. Meanwhile, Ang II treatment induced F-actin polymerization, which was inhibited after ROCK inhibition. In mice, Ang II infusion increased VSMC migration into the neointima and reduced VSMC differentiation proteins levels, and these effects were shown to be dependent on AT1 receptor and RhoA/ROCK pathway. CONCLUSION: This study reveals a novel mechanism by which Ang II regulates RhoA/ROCK signaling and actin polymerization via AT1 receptor and then affects VSMC dedifferentiation.

The multiple sclerosis (MS) drugs as a potential treatment of ARDS in COVID-19 patients.

We encourage studies on the effectiveness of multiple sclerosis drugs for the treatment of ARDS in COVID-19 infection. These drugs, through the inhibition of the RhoA/actin-dependent expression of virus receptors in the macrophages and macrophage recruitment to the lungs, have the potential to inhibit cytokine storm of lung macrophages, reduce or eliminate ARDS and improve the outcome of COVID-19 infection.

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

3,3'-Diindolylmethane modulates aryl hydrocarbon receptor of esophageal squamous cell carcinoma to reverse epithelial-mesenchymal transition through repressing RhoA/ROCK1-mediated COX2/PGE2 pathway.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive tumors in the world. Aryl hydrocarbon receptor (AHR) has been reported to promote tumor metastasis and epithelial-mesenchymal transition (EMT) is a vital process of conferring cancer cells capabilities of migration and invasion. However, the mechanism by which modulation of AHR can inhibit tumor metastasis remains unknown. Thus, we aim to investigate the underlying mechanism regarding reversing EMT process of ESCC through modulation of AHR. METHODS: We used AHR selective modulator 3,3'-diindolylmethane (DIM) to treat ESCC cell lines TE1 and KYSE150 so as to examine alterations of migration and invasion by wound healing and Transwell assay. Western blotting (WB) and qPCR were performed to detect relative genes and proteins changes regarding EMT process. Cell transfection was utilized for confirming pathways involved in DIM-induced reversal of EMT and in vivo assay was conducted for verification of the underlying mechanism. Co-IP assay was conducted for detecting protein-protein interactions. RESULTS: AHR was overexpressed in ESCC and modulation of AHR by DIM could inhibit migration and invasion as well as downregulate mesenchymal cell markers ß-Catenin, Vimentin and Slug and upregulate epithelial cell marker Claudin-1. Meanwhile, synergically overexpression of AHR, RhoA and ROCK1 correlated with poor clinical outcomes. DIM could inhibit COX2/PGE2 pathway by targeting AHR, and COX2 selective inhibitor Celecoxib could suppress EMT and metastasis. Results of PGE2 treatment were opposite to that of Celecoxib. Meanwhile, blockade of RhoA/ROCK1 pathway also exerted prohibitive effects on EMT and metastasis. WB results showed COX2/PGE2 pathway could be regulated by RhoA/ROCK1 pathway and DIM could inhibit RhoA/ROCK1 pathway through modulation of AHR. In vivo assay verified the results in vitro. Co-IP results showed DIM could modulate AHR to reverse EMT directly through inhibition of interaction between AHR and EGFR (epidermal growth factor receptor) so as to block RhoA/ROCK1-mediated COX2/PGE2 pathway which was connected by NF-κB. CONCLUSIONS: In brief, modulation of AHR by DIM can reverse EMT process and inhibit metastasis of ESCC through repressing RhoA/ROCK1-mediated COX2/PGE2 pathway.

The influence of hypoxia and energy depletion on the response of endothelial cells to the vascular disrupting agent combretastatin A-4-phosphate.

Combretastatin A-4 phosphate (CA4P) is a microtubule-disrupting tumour-selective vascular disrupting agent (VDA). CA4P activates the actin-regulating RhoA-GTPase/ ROCK pathway, which is required for full vascular disruption. While hypoxia renders tumours resistant to many conventional therapies, little is known about its influence on VDA activity. Here, we found that active RhoA and ROCK effector phospho-myosin light chain (pMLC) were downregulated in endothelial cells by severe hypoxia. CA4P failed to activate RhoA/ROCK/pMLC but its activity was restored upon reoxygenation. Hypoxia also inhibited CA4P-mediated actinomyosin contractility, VE-cadherin junction disruption and permeability rise. Glucose withdrawal downregulated pMLC, and coupled with hypoxia, reduced pMLC faster and more profoundly than hypoxia alone. Concurrent inhibition of glycolysis (2-deoxy-D-glucose, 2DG) and mitochondrial respiration (rotenone) caused profound actin filament loss, blocked RhoA/ROCK signalling and rendered microtubules  CA4P-resistant. Withdrawal of the metabolism inhibitors restored the cytoskeleton and CA4P activity. The AMP-activated kinase AMPK was investigated as a potential mediator of pMLC downregulation. Pharmacological AMPK activators that generate AMP, unlike allosteric activators, downregulated pMLC but only when combined with 2DG and/or rotenone. Altogether, our results suggest that Rho/ROCK and actinomyosin contractility are regulated by AMP/ATP levels independently of AMPK, and point to hypoxia/energy depletion as potential modifiers of CA4P response.

Ursolic acid reverses liver fibrosis by inhibiting interactive NOX4/ROS and RhoA/ROCK1 signalling pathways.

Liver fibrosis is the reversible deposition of extracellular matrix (ECM) and scar formation after liver damage by various stimuli. The interaction between NOX4/ROS and RhoA/ROCK1 in liver fibrosis is not yet clear. Ursolic acid (UA) is a traditional Chinese medicine with anti-fibrotic effects, but the molecular mechanism underlying these effects is still unclear. We investigated the interaction between NOX4/ROS and RhoA/ROCK1 during liver fibrosis and whether these molecules are targets for the anti-fibrotic effects of UA. First, we confirmed that UA reversed CCl4-induced liver fibrosis. In the NOX4 intervention and RhoA intervention groups, related experimental analyses confirmed the decrease in CCl4-induced liver fibrosis. Next, we determined that the expression of NOX4 and RhoA/ROCK1 was decreased in UA-treated liver fibrotic mice. Furthermore, RhoA/ROCK1 expression was decreased in the NOX4 intervention group, but there was no significant change in the expression of NOX4 in the RhoA intervention group. Finally, we found that liver fibrotic mice showed a decline in their microbiota diversity and abundance, a change in their microbiota composition, and a reduction in the number of potential beneficial bacteria. However, in UA-treated liver fibrotic mice, the microbiota dysbiosis was ameliorated. In conclusion, the NOX4/ROS and RhoA/ROCK1 signalling pathways are closely linked to the development of liver fibrosis. UA can reverse liver fibrosis by inhibiting the NOX4/ROS and RhoA/ROCK1 signalling pathways, which may interact with each other.