Downstream Effector Of RhoA Research Articles

Novel Small-Molecule ROCK2 Inhibitor GNS-3595 Attenuates Pulmonary Fibrosis in Preclinical Studies.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that leads to respiratory decline caused by scarring and thickening of lung tissues. Multiple pathways contribute to the fibrotic process in this disease, such as inflammation, epithelial-to-mesenchymal transition, and oxidative stress. The Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway is a key regulator of profibrotic signaling, as it affects the organization of actin-myosin and the remodeling of the extracellular matrix. ROCK1/2, a downstream effector of RhoA, is overexpressed in patients with IPF and is a promising target for IPF therapy. However, because of the hypotensive side effects of ROCK1/2 inhibitors, selective ROCK2 compounds are being explored. In this study, we report the discovery of GNS-3595, a potent and selective ROCK2 inhibitor that has ∼80-fold selectivity over ROCK1 at physiological concentrations of ATP. GNS-3595 effectively inhibited ROCK2-mediated phosphorylation of myosin light chain and reduced the expression of fibrosis-related proteins (e.g., collagen, fibronectin, and α-smooth muscle actin) in various invitro cellular models. GNS-3595 also prevented transforming growth factor β-induced fibroblast-to-myofibroblast transition. In addition, in a bleomycin-induced mouse model of pulmonary fibrosis, therapeutic exposure to GNS-3595, suppressed lung fibrosis, stabilized body weight loss, and prevented fibrosis-induced lung weight gain. Transcriptome and protein expression analysis from lung tissues showed that GNS-3595 can revert the fibrosis-related gene expression induced by bleomycin. These results indicate that GNS-3595 is a highly potent, selective, and orally active ROCK2 inhibitor with promising therapeutic efficacy against pulmonary fibrosis.

Insights into Calpain Activation and Rho-ROCK Signaling in Parkinson's Disease and Aging.

Parkinson's disease (PD), a progressive neurodegenerative disease, has no cure, and current therapies are not effective at halting disease progression. The disease affects mid-brain dopaminergic neurons and, subsequently, the spinal cord, contributing to many debilitating symptoms associated with PD. The GTP-binding protein, Rho, plays a significant role in the cellular pathology of PD. The downstream effector of Rho, Rho-associated kinase (ROCK), plays multiple functions, including microglial activation and induction of inflammatory responses. Activated microglia have been implicated in the pathology of many neurodegenerative diseases, including PD, that initiate inflammatory responses, leading to neuron death. Calpain expression and activity is increased following glial activation, which triggers the Rho-ROCK pathway and induces inflammatory T cell activation and migration as well as mediates toxic α-synuclein (α-syn) aggregation and neuron death, indicating a pivotal role for calpain in the inflammatory and degenerative processes in PD. Increased calpain activity and Rho-ROCK activation may represent a new mechanism for increased oxidative damage in aging. This review will summarize calpain activation and the role of the Rho-ROCK pathway in oxidative stress and α-syn aggregation, their influence on the neurodegenerative process in PD and aging, and possible strategies and research directions for therapeutic intervention.

Proximal-tubule molecular relay from early Protein diaphanous homolog 1 to late Rho-associated protein kinase 1 regulates kidney function in obesity-induced kidney damage

The small GTPase protein RhoA has two effectors, ROCK (Rho-associated protein kinase 1) and mDIA1 (protein diaphanous homolog 1), which cooperate reciprocally. However, temporal regulation of RhoA and its effectors in obesity-induced kidney damage remains unclear. Here, we investigated the role of RhoA activation in the proximal tubules at the early and late stages of obesity-induced kidney damage. In mice, a three-week high-fat-diet induced proximal tubule hypertrophy and damage without increased albuminuria, and RhoA/mDIA1 activation without ROCK activation. Conversely, a 12-week high-fat diet induced proximal tubule hypertrophy, proximal tubule damage, increased albuminuria, and RhoA/ROCK activation without mDIA1 elevation. Proximal tubule hypertrophy resulting from cell cycle arrest accompanied by downregulation of the multifunctional cyclin-dependent kinase inhibitor p27Kip1 was elicited by RhoA activation. Mice overexpressing proximal tubule-specific and dominant-negative RHOA display amelioration of high-fat diet-induced kidney hypertrophy, cell cycle abnormalities, inflammation, and renal impairment. In human proximal tubule cells, mechanical stretch mimicking hypertrophy activated ROCK, which triggered inflammation. In human kidney samples from normal individuals with a body mass index of about 25, proximal tubule cell size correlated with body mass index, proximal tubule cell damages, and mDIA1 expression. Thus, RhoA activation in proximal tubules is critical for the initiation and progression of obesity-induced kidney damage. Hence, the switch in the downstream RhoA effector in proximal tubule represents a transition from normal to pathogenic kidney adaptation and to body weight gain, leading to obesity-induced kidney damage.

Role of Rho-Associated Protein Kinase Inhibition As Therapeutic Strategy for Parkinson's Disease: Dopaminergic Survival and Enhanced Mitophagy.

The GTP-binding protein, Rho, plays a significant role in the cellular pathology of Parkinson’s disease. The downstream effector of Rho, Rho-associated kinase (ROCK), performs several functions, including microglial inflammatory response and enhanced Parkin-mediated mitophagy. Its inhibition shows neuroprotective effects in carried studies. Parkinson’s disease pathology also rests on incomplete removal of damaged mitochondria, leading to neuronal impairment. ROCK has different isoforms, inhibition of which have been shown to decrease the adverse changes in microglia. There has also been evidence of a decreased release of inflammatory cytokines and a reduction in degradation of dopaminergic neurons on the addition of ROCK inhibitors. Additionally, ROCK inhibitors have recently been shown to increase the activity of hexokinase 2 (HK2), relocating it to mitochondria, and therefore leading to upregulated mitochondrial targeting. Understanding the cellular basis of ROCK activity and its inhibition may help us advance in creating new strategies for the treatment of Parkinson’s disease.

ROCK (RhoA/Rho Kinase) in Cardiovascular-Renal Pathophysiology: A Review of New Advancements.

Rho-associated, coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase and found to belong to the AGC family of serine/threonine kinases. They were shown to be downstream effectors of RhoA and RhoC activation. They signal via phosphorylation of proteins such as MYPT-1, thereby regulating many key cellular functions including proliferation, motility and viability and the RhoA/ROCK signaling has been shown to be deeply involved in arterial hypertension, cardiovascular–renal remodeling, hypertensive nephropathy and posttransplant hypertension. Given the deep involvement of ROCK in cardiovascular–renal pathophysiology and the interaction of ROCK signaling with other signaling pathways, the reports of trials on the clinical beneficial effects of ROCK’s pharmacologic targeting are growing. In this current review, we provide a brief survey of the current understanding of ROCK-signaling pathways, also integrating with the more novel data that overall support a relevant role of ROCK for the cardiovascular–renal physiology and pathophysiology.

RhoA Targeting Suppresses Cold-Induced Platelet Lesion and Allows Refrigerated Storage of Functional Human Platelets to Fourteen Days

Platelet transfusion is required for the support therapy of patients with hematological disorders and cancer. Current practice of donor-derived platelet storage at room temperature (RT) associates with an inherent risk of microbial contamination and a limit of 5-7 day shelf life. Refrigerated platelets are hemostatically superior than RT platelets but their survival in circulation is severely reduced. Storage in cold temperature induces loss of galactosylation/sialylation of platelet GPIb with exposure of b-N-acetyl glucosamine of N-linked glycans, which clusters on the platelet plasma membrane. Upon transfusion, these clusters are recognized by macrophages and hepatocytes resulting in lectin-mediated platelet clearance. We hypothesized that the long-term refrigerated storage lesion depends on the mislocalization of membrane-bound glycosyl- and syalyl-transferases in lipid rafts and endocytotic intermediates of cold-stored platelets. By using a combination of genetic and pharmacological approaches, we investigated whether the three major members of the Rho GTPase family, RHOA, RAC1 and/or CDC42, which are molecular switches regulating actomyosin dynamics and signaling in platelets. We found that cooling of platelets induces a sustained activation of RhoA and Rac1, but not Cdc42 in mouse and human platelets. Inducible genetic deletion of platelet RhoA in mice prevents clearance of cold-stored platelets upon transfusion in wild-type congenic animals (WT: 2±0.5 hrs x fraction of infusate; RhoAΔ/Δ: 8± 0.7 hrs x fraction of infusate; p≤0.05). When human platelets were stored in either plasma or PAS-IIIM (PAS-E) for up to 14 days, the inclusion in the storage medium of a rationally designed RHOA inhibitor G04 can prevent the phagocytosis of 14-day refrigerated platelets by activated macrophages in vitro (RT-stored, vehicle-treated platelets: 20%±4; cold stored, vehicle-treated 70±5%; cold-treated, G04(10μM)- treated: 25±3%; p≤0.001) or in vivo clearance as assessed by the area under the curve of surviving human platelets (hrs x fraction of infusate) transfused into clodronate-treated, sub-lethally irradiated non-obese diabetic, γc-/- (NSG) mice (RT-stored, vehicle-treated platelets: 16±1.0; cold-stored, vehicle-treated: 6±0.5; cold-stored, G04(10μM)-treated: 15±0.6; p≤0.001). This inhibition is reversible by either a wash or a 3-fold dilution that resulted in a reversion of the inhibitory effect on aggregation and in vivo correction of the bleeding time of mice pre-treated with aspirin (RT-stored, vehicle-treated: 45±4 sec.; cold-stored, vehicle-treated: 150±10 sec.; cold-stored, G04(10μM)-treated: 76±3.8 sec.; p≤0.001). Mechanistically, RHOA inhibition prevents the cold-induced cytoskeleton/shape change and spreading on fibrinogen through the prevention of the formation of lipid rafts enriched in glycosyl- and syalyl-transferase activities and the endocytosis of vacuoles enriched in GPIbα. Addition of the lipid raft disruptor b-cyclodextrin, but not of the RHOA downstream effector ROCK inhibitor Fasudil, phenocopies the effect of G04 on the prevention of cold-induced platelet damage. Thus, RHOA is the key mediator of the platelet storage lesion through a ROCK independent mechanism and its reversible inhibition allows the functional maintenance of cold-stored platelets in both survival and hemostatic properties. Our pre-clinical data support the concept that a platelet additive solution containing a low-affinity RHOA inhibitor is useful in preventing platelet storage lesion while fully maintaining their hemostatic function after 14 days of storage. A rationally designed cold storage regimen is highly feasible, which could resolve the platelet clearance problem and meet an urgent need in transfusion medicine for the support therapy of patients with thrombocytopenia or thrombocytopathy. Disclosures Cancelas: Cellphire: Research Funding; Velico: Consultancy, Research Funding; Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding.

Silver nanoparticles promote osteogenic differentiation of human periodontal ligament fibroblasts by regulating the RhoA-TAZ axis.

Silver nanoparticles (AgNps) are well established antibacterial agents, which have been reported to promote osteogenesis of stem cells. Ras homolog gene family member A (RhoA) and Tafazzin (TAZ) have been recently shown to be involved in osteogenic differentiation. The present study aimed to evaluate the effects of AgNps on osteogenic differentiation of human periodontal ligament fibroblasts (HPDLFs), and investigate the underlying mechanisms of AgNps activity. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to determine the safe concentration of AgNps in HPDLFs. Using an alkaline phosphatase assay, Alizarin red S staining and detection of the expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osterix (OSX), and collagen-I, we found that AgNps, at a concentration range of 25-100 μM, promoted osteogenic differentiation of HPDLFs in a dose-dependent manner. We also found that 100 μM AgNps up-regulated the active RhoA expression level. The results of ALP activity and expression of osteogenic markers showed that the effects of AgNps on osteogenic differentiation were abrogated by a RhoA pathway inhibitor, C3 reagent. Additionally, silencing of TAZ attenuated the AgNps-induced osteogenic differentiation of HPDLFs, as shown by decreased ALP activity and down-regulation of osteogenic markers. Interestingly, TAZ knockdown had a very small effect on the activity of RhoA, whereas C3 suppressed the expression of TAZ, indicating that TAZ was a downstream effector of RhoA. In conclusion, the present work demonstrated that AgNps promoted osteogenic differentiation of HPDLFs by activating the RhoA-TAZ axis.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting indisruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential forglaucoma, the leading cause of irreversible blindness in humans.

Limitation by Rho-kinase and Rac of transforming growth factor-β-induced interleukin-6 release from astrocytes

Transforming growth factor (TGF)-β stimulates release of interleukin (IL)-6, which is recognized to function as both a pro- and anti- inflammatory cytokine in the central nervous system, from astrocytes. It is generally recognized that effects of TGF-β are mediated through Smad-independent as well as Smad-dependent pathways. Small GTPases regulate a variety of cell functions. In the present study, we investigated whether or not Rho-kinase, a downstream effector of Rho, and Rac are implicated in TGF-β-stimulated IL-6 release from astrocytes (C8D1A cells). Y-27632 or fasudil (Rho-kinase inhibitors) or NSC23766 (an inhibitor of Rac-guanine nucleotide exchange factor interaction) significantly enhanced TGF-β-stimulated IL-6 release from these cells. TGF-β-stimulated IL-6 release was markedly upregulated in RhoA- or Rac-knockdown C8D1A cells. We found that SIS3 (a specific inhibitor of TGF-β-dependent Smad3 phosphorylation) or LY364947 (a TGF-β type I receptor kinase inhibitor) significantly reduced the IL-6 release. However, TGF-β-induced-Smad2 and Smad3 phosphorylation was not affected by Y-27632, fasudil or NSC23766. In conclusion, our results strongly suggest that Rho-kinase and Rac limit TGF-β-induced IL-6 release from astrocytes, and the suppressive effects are exerted independently of the Smad pathway or at a point downstream of Smad2/3 complex.

MDia1 and Cdc42 Regulate Activin B-Induced Migration of Bone Marrow-Derived Mesenchymal Stromal Cells.

In a previous study, we have shown that Activin B is a potent chemoattractant for bone marrow‐derived mesenchymal stromal cells (BMSCs). As such, the combination of Activin B and BMSCs significantly accelerated rat skin wound healing. In another study, we showed that RhoA activation plays a key role in Activin B‐induced BMSC migration. However, the role of the immediate downstream effectors of RhoA in this process is unclear. Here, we demonstrated that mammalian homolog of Drosophila diaphanous‐1 (mDia1), a downstream effector of RhoA, exerts a crucial function in Activin B‐induced BMSC migration by promoting membrane ruffling, microtubule morphology, and adhesion signaling dynamics. Furthermore, we showed that Activin B does not change Rac1 activity but increases Cdc42 activity in BMSCs. Inactivation of Cdc42 inhibited Activin B‐stimulated Golgi reorientation and the cell migration of BMSCs. Furthermore, knockdown of mDia1 affected Activin B‐induced BMSC‐mediated wound healing in vivo. In conclusion, this study demonstrated that the RhoA‐mDia1 and Cdc42 pathways regulate Activin B‐induced BMSC migration. This study may help to optimize clinical MSC‐based transplantation strategies to promote skin wound healing. stem cells 2019;37:150–161

RhoA regulates Schwann cell differentiation through JNK pathway

RhoA is a small GTPase that regulates many functions of mammalian cells via actin reorganization. Lots of studies uncovered that its activation acts as a major negative regulator of neurite extension, and inhibition of RhoA activity or reduction of its expression can promote neuron survival and axonal regeneration. However, little is known about whether RhoA also exerts important functions on Schwann cells (SCs) which are the glial cells of the peripheral nervous system (PNS). Recently, we reported that RhoA plays important roles in the proliferation, migration and myelination of SCs. In the present study, using RNA interference to knockdown RhoA expression and CT04 (a cell-permeable C3 Transferase) to inhibit RhoA activation we found that blocking RhoA can slack SC differentiation. Unexpectedly, inhibiting ROCK, the mostly well-known downstream effector of RhoA, has no influence on SC differentiation. Instead, the inhibition of RhoA in differentiating SCs results in the activation of JNK and p38 MAPK. And the inhibitor of JNK but not p38 MAPK can promote SC differentiation in the presence of RhoA inhibition. Overall results indicate that RhoA plays a vital role in SC differentiation via JNK pathway rather than ROCK pathway.

Rho Kinase Type 1 (ROCK1) Promotes Lipopolysaccharide-induced Inflammation in Corneal Epithelial Cells by Activating Toll-Like Receptor 4 (TLR4)-Mediated Signaling.

BackgroundRho kinases (ROCKs) are the typical downstream effectors of RhoA, which can regulate corneal epithelial wound healing. In this study, the role of ROCK1 in lipopolysaccharide (LPS)-induced cornea inflammation was investigated.Material/MethodsThe expression of ROCK1 in human corneal epithelial cells (HCECs) was bilaterally modulated with ROCK1 expression vector and ROCK1 inhibitor Y-27632. The effects of ROCK1 modulation on the inflammatory response, cell viability, cell apoptosis, and cell cycle distribution were detected by ELISA assay, MTT assay, and flow cytometry, respectively. The pathways involved in the effect of ROCK1 in HCECs was preliminarily explained by detecting changes of TLR4-mediated NF-κB and ERK signaling using western blotting and electrophoretic mobility shift assays.ResultsOverexpression of ROCK1 promoted LPS-induced production of IL-6, IL-8, IL-1β, and TNF-α, and the apoptotic process in HCECs. Augmented inflammation and apoptosis were associated with stronger activation of TLR4-mediated signal transduction; the phosphorylation of IκBα, JNK, ERK1/2, and p38, and nuclear translocation of NF-κB p65 induced by LPS were further increased by overexpression of ROCK1. Inhibition of ROCK1 function by Y-27632 blocked the effect of LPS on HCECs; both LPS-induced inflammation and apoptosis was alleviated by Y-27632, which was associated with suppression of TLR4-mediated NF-κB and ERK signaling.ConclusionsLPS-induced inflammation and apoptosis in HCECs depended on the function of ROCK1, inhibition of which would attenuate impairments on cornea cells due to LPS.

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.

Direct visualization and light control of several cellular processes is a challenge due to spectral overlap of available genetically-encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein miRFP720 and the fully NIR Förster Resonance Energy Transfer (FRET) pair miRFP670-miRFP720 that enables design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use for multiplexed imaging and light control of RhoGTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and then concurrently used LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified the antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK, showed that Rac1 activity and GDI binding depend exquisitely on the spatiotemporal coordination between these two molecules, and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.

Pseudophosphatase MK‐STYX regulates neurite outgrowth and alters the morphology of primary neurons

Mitogen‐activated protein kinases (MAPKs) are essential players in important neuronal signaling pathways including neuronal development, plasticity, survival, learning, and memory. The inactivation of MAPKs is tightly controlled by MAPK phosphatases (MKPs), which also are important regulators of these neuronal processes. There is compelling evidence that a unique catalytically inactive member of the MKP family, MK‐STYX (mitogen activated kinase phosphoserine/threonine/tyrosine binding protein), is a regulator in neuronal processes such as neurite outgrowth. We previously reported that the MK‐STYX dramatically increases the number of primary neurites in rat pheochromocytoma (PC‐12) cells through the RhoA signaling pathway. MK‐STYX decreases RhoA activation, whereas knockdown of MK‐STYX increases RhoA activation. Furthermore, MK‐STYX increases phosphorylation of cofilin, a RhoA downstream effector, whereas phosphorylated cofilin decreases when MK‐STYX is knocked down with shRNA. Throughout the course of these experiments, we noticed that cells overexpressing MK‐STYX developed more neurites, which often branched. Here, we report that microtubules and microfilaments, components of the cytoskeleton that are involved in the formation of neurites, are present in MK‐STYX‐induced outgrowths. In addition, in response to nerve growth factor (NGF), MK‐STYX‐producing cells produced more actin growth cones than non‐MK‐STYX‐expressing cells. Transmission electron microscopy confirmed that MK‐STYX‐induced neurites form synapses. Furthermore, the expression of Tau‐1 and MAP2 (microtubule associated protein 2) in MK‐STYX‐induced neurites suggests that they have both axonal and dendritic properties. Further studies in hippocampal primary neurons demonstrated that MK‐STYX altered their morphology. A significant number of primary neurons expressing MK‐STYX had more than the normal number of primary neurites. Taken together these data provide evidence that MK‐STYX causes cytoskeletal rearrangement. It also highlights that this unique member of the MKP subfamily has the potential to have a major role in neuronal signaling.Support or Funding InformationThis work was supported by the National Science Foundation Grant MCB1113167 to S.D.H.; Coco Award to S.D.H.; Howard Hughes Medical Research Institute grant through the Undergraduate Science Education Program to the College of William and Mary; a DuPont Fund Student‐Faculty Research Team Stipend awarded to A.D. and S.D.H. through a grant by the Jessie Ball Grant Fund to the William and Mary Scholars for Undergraduate Research Experience program; Ferguson Awards to D.A.B., A.D., and C.S., and the Dintersmith Honors Fellowship to A.D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of the clinical use of ROCK inhibitor on the pathogenesis and treatment of glaucoma.

Rho-associated protein kinase (ROCK), a ubiquitously expressed signaling messenger and downstream effector of Rho, is activated by several bioactive factors in the aqueous humor (AH). Rho-ROCK signaling regulates a wide spectrum of fundamental cellular events, including cell adhesion, motility, proliferation, differentiation, and apoptosis. Previous studies, including our own, found that ROCK inhibitor lowers intraocular pressure (IOP) via a direct effect on the conventional AH outflow pathway, by regulation of contractile properties, fibrotic activity, and permeability of the trabecular meshwork (TM) and Schlemm's canal (SC) tissues, influencing extracellular matrix (ECM) production. Recently, a novel ROCK inhibitor, ripasudil, has been introduced in Japan. Other ROCK inhibitors are now in clinical trials as new IOP-lowering drugs for glaucoma patients. To date, ripasudil, administered together with other glaucoma medications, has proved safe and efficient in lowering IOP as well as additional effects such as prostaglandin analogs, beta-blockers, and carbonic anhydrase inhibitors, all of which help lower IOP by different mechanisms. In addition, we found that long-term treatment with ripasudil exerted an additional IOP-lowering effect, especially in eyes with high IOP, suggesting that late-onset remodeling of the ECM in glaucomatous eyes may elicit mild and delayed changes in IOP levels. ROCK inhibitors have also shown several additional effects, including increased retinal blood flow, direct protection of neurons against various types of stress, and regulation of wound healing; these benefits may potentially be useful in glaucoma treatment.

Translational Advances in the Management of Acute Spinal Cord Injury

Translational Advances in the Management of Acute Spinal Cord Injury

C‑Maf inducing protein inhibits cofilin‑1 activity and alters podocyte cytoskeleton organization.

The glomerular visceral epithelial cells, also termed podocytes, are key in maintaining the normal renal filtration barrier. Although it has been demonstrated that stimulation of c‑Maf inducing protein (CMIP) expression is involved in podocyte damage, the molecular events during this process remain unclear. In the current study, CMIP‑induced proximal signaling was investigated by focusing on its effect on cofilin‑1 activity in puromycin aminonucleoside (PA)‑damaged podocytes. An obvious elevation of CMIP expression and phosphorylated (p) cofilin‑1 levels was detected in cultured podocytes treated with PA and in glomeruli isolated from PA‑induced nephropathy rats. Stable knockdown of CMIP prevented upregulation of p‑cofilin‑1 and reorganization of actin cytoskeleton in PA‑treated podocytes. The activity of the Src family kinase Fyn was reduced, whereas small GTPase Ras homolog gene family, member A (RhoA) activity was increased in PA‑treated podocytes. Stimulation of CMIP expression inhibited Fyn activation and decreased the expression level of p‑p190RhoGAP, a negative regulator of RhoA activity. The level of p‑LIM domain kinase 1 (LIMK1), a downstream effector of RhoA, increased significantly in PA‑treated podocytes. Notably, the applications of RhoA inhibitor or knockdown of LIMK prevented increase of the p‑cofilin‑1 level in PA‑treated podocytes. Thus, the current data provided evidence that the CMIP/Fyn/RhoA/cofilin‑1 signaling pathway may be associated with actin disorganization and podocyte foot process spreading following podocyte injury.

ROCK1 is a novel Rac1 effector to regulate tubular endocytic membrane formation during clathrin-independent endocytosis

Clathrin-dependent and -independent pathways contribute for β1-integrin endocytosis. This study defines a tubular membrane clathrin-independent endocytic network, induced with the calmodulin inhibitor W13, for β1-integrin internalization. This pathway is dependent on increased phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) levels and dynamin activity at the plasma membrane. Exogenous addition of PI(4,5)P2 or phosphatidylinositol-4-phosphate 5-kinase (PIP5K) expression mimicked W13-generated-tubules which are inhibited by active Rac1. Therefore, the molecular mechanisms downstream of Rac1, that controls this plasma membrane tubulation, were analyzed biochemically and by the expression of different Rac1 mutants. The results indicate that phospholipase C and ROCK1 are the main Rac1 effectors that impair plasma membrane invagination and tubule formation, essentially by decreasing PI(4,5)P2 levels and promoting cortical actomyosin assembly respectively. Interestingly, among the plethora of proteins that participate in membrane remodeling, this study revealed that ROCK1, the well-known downstream RhoA effector, has an important role in Rac1 regulation of actomyosin at the cell cortex. This study provides new insights into Rac1 functioning on plasma membrane dynamics combining phosphatidylinositides and cytoskeleton regulation.

Effects of chronic Δ9-tetrahydrocannabinol treatment on Rho/Rho-kinase signalization pathway in mouse brain

Δ9-Tetrahydrocannabinol (Δ9-THC) shows its effects by activating cannabinoid receptors which are on some tissues and neurons. Cannabinoid systems have role on cell proliferation and development of neurons. Furthermore, it is interesting that cannabinoid system and rho/rho-kinase signalization pathway, which have important role on cell development and proliferation, may have role on neuron proliferation and development together. Thus, a study is planned to investigate rhoA and rho-kinase enzyme expressions and their activities in the brain of chronic Δ9-THC treated mice. One group of mice are treated with Δ9-THC once to see effects of acute treatment. Another group of mice are treated with Δ9-THC three times per day for one month. After this period, rhoA and rho-kinase enzyme expressions and their activities in mice brains are analyzed by ELISA method. Chronic administration of Δ9-THC decreased the expression of rhoA while acute treatment has no meaningful effect on it. Administration of Δ9-THC did not affect expression of rho-kinase on both chronic and acute treatment. Administration of Δ9-THC increased rho-kinase activity on both chronic and acute treatment, however, chronic treatment decreased its activity with respect to acute treatment. This study showed that chronic Δ9-THC treatment down-regulated rhoA expression and did not change the expression level of rho-kinase which is downstream effector of rhoA. However, it elevated the rho-kinase activity. Δ9-THC induced down-regulation of rhoA may cause elevation of cypin expression and may have benefit on cypin related diseases. Furthermore, use of rho-kinase inhibitors and Δ9-THC together can be useful on rho-kinase related diseases.

Reciprocal regulation of Th17 and Treg cell differentiation by RhoA

Abstract Th17 cells are important for protective immunity as well as tissue inflammation and autoimmune disorders. Treg cells, including induced Treg (iTreg) and natural Treg (nTreg), play a critical role in immune tolerance. RhoA of the Rho small GTPase family is an intracellular signal transducer that cycles between an inactive GDP-bound form and an active GTP-bound form. We have previously reported that RhoA plays an important role in Th2 cell differentiation by regulating glycolysis. In this study, we further examined the role of RhoA in Th17 and Treg cell differentiation. Genetic deletion of RhoA in T cells or pharmacologic inhibition of RhoA by a RhoA-specific inhibitor Y16 led to reduced Th17 differentiation in vitro as evidenced by reduction of Th17 effector cytokines, transcriptional downregulation of RORγT and reduced phosphorylation of Stat3. Accordingly, RhoA deficiency ameliorated house dust mites (HDM)-induced allergic airway inflammation and dextran sodium sulphate (DSS)-induced colitis in vivo where Th17 cells plays an important role. Inhibition of ROCK, an immediate downstream effector of RhoA, recapitulated the effect of RhoA deficiency or inhibition on Th17 differentiation, suggesting that RhoA regulates Th17 cell differentiation through ROCK. On the other hand, ablation of RhoA had no effect on iTreg cell differentiation. However, inhibition of ROCK enhanced iTreg differentiation, suggesting that ROCK has RhoA-independent role in iTreg differentiation. In contrast to its in-effect on iTreg differentiation, RhoA deficiency increased nTreg differentiation. Thus, our data have provided new insights into Th17 and Treg differentiation and identified RhoA as a potential therapeutic target for tissue inflammation.