Neuropeptide orphanin FQ inhibits dendritic morphogenesis through activation of RhoA

Brain‐derived neurotrophic factor (BDNF) plays a facilitatory role in neuronal development and promotion of differentiation. Mechanisms that oppose BDNF's stimulatory effects create balance and regulate dendritic growth. However, these mechanisms have not been studied. We have focused our studies on the BDNF‐induced neuropeptide OrphaninFQ/ Nociceptin (OFQ); while BDNF is known to enhance synaptic activity, OFQ has opposite effects on activity, learning, and memory. We have now examined whether OFQ provides a balance to the stimulatory effects of BDNF on neuronal differentiation in the hippocampus. Golgi staining in OFQ knockout (KO) mice revealed an increase in primary dendrite length as well as spine density, suggesting that endogenous OFQ inhibits dendritic morphology. We have also used cultured hippocampal neurons to demonstrate that exogenous OFQ has an inhibitory effect on dendritic growth and that the neuropeptide alters the response to BDNF when pre‐administered. To determine if BDNF and OFQ act in a feedback loop, we inhibited the actions of the BDNF and OFQ receptors, TrkB and NOP using ANA‐12 and NOP KO mice respectively but our data suggest that the two factors do not act in a negative feedback loop. We found that the inhibition of dendritic morphology induced by OFQ is via enhanced RhoA activity. Finally, we have evidence that RhoA activation is required for the inhibitory effects of OFQ on dendritic morphology. Our results reveal basic mechanisms by which neurons not only regulate the formation of proper dendritic growth during development but also control plasticity in the mature nervous system. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 769–784, 2013     

Carboxy-terminal fragment of osteogenic growth peptide regulates myeloid differentiation through RhoA

The carboxy-terminal fragment of osteogenic growth peptide, OGP(10-14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10-14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10-14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10-14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10-14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out.     

Redox regulation of RhoA

We have previously shown that redox agents including superoxide anion radical and nitrogen dioxide can react with GXXXXGK(S/T)C motif-containing GTPases (i.e., Rac1, Cdc42, and RhoA) to stimulate guanine nucleotide release. We now show that the reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the RhoA redox-active motif. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to downregulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity. The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, the results presented herein suggest that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA to produce a RhoA thiol-cisplatin-thiol adduct, leading to inactivation of RhoA. Similarly, certain arsenic complexes (i.e., arsenate and arsenic trioxide) may inactivate RhoA by bridging the cysteine residues in the GXXXCGK(S/T)C motif. Thus, in addition to redox agents, platinated-chemotherapeutic agents and arsenic complexes may modulate the activity of GTPases containing the GXXXCGK(S/T)C motif (i.e., RhoA and RhoB).     

Nuanced junctional RhoA activity

RhoA signalling controls many diverse cellular processes, and thus discovering the mechanisms that determine its specific outcomes is a tantalizing challenge. A previously uncharacterized regulatory module operates selectively at the zonula adherens of epithelial cell junctions, in which positive and negative RhoA regulators are coordinated to fine-tune RhoA activity.     

Akt and RhoA inhibition promotes anoikis of aggregated B16F10 melanoma cells

In the highly metastatic B16F10 melanoma cell line, activation of the signalling molecules that promote cell proliferation and survival on conventional adhesive culture dishes may also be responsible for the growth and resistance to anoikis of aggregates on a non-adhesive substratum. We have examined the influence of bacterial ADP-ribosyltransferases C3-like exoenzymes, which selectively modify RhoA, B and C proteins and inhibit signal pathways controlled by them. RNA interference [siRNA (small interfering RNA) Akt (also known as protein kinase B)] and a PI3K (phosphoinositide 3-kinase) inhibitor were used to analyse the changes caused by inhibiting the PI3K/Akt pathway. Inhibiting the activation of RhoA, B, C and Akt expression resulted in a decrease of the number of cells cultured in aggregates, and caspase 3 activation. RhoA activation and RhoB and RhoC expression were controlled by Akt, but not RhoA expression. Inhibiting Akt and RhoA reduced the expression of α5 integrin, and inactivated FAK (focal adhesion kinase) in B16F10 cells cultured as aggregates. Thus, inhibiting Rho subfamily proteins and Akt expression inactivates the FAK pathway and induces anoikis in anoikis-resistant cells. The activation of RhoA in melanoma cells can depend on PI3K/Akt activation, suggesting that PI3K/Akt is a suitable target for new therapeutic approaches.     

Targeting by myosin phosphatase-RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase

Vascular smooth muscle cell contractile state is the primary determinant of blood vessel tone. Vascular smooth muscle cell contractility is directly related to the phosphorylation of myosin light chains (MLCs), which in turn is tightly regulated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase. Myosin phosphatase is the principal enzyme that dephosphorylates MLCs leading to relaxation. Myosin phosphatase is regulated by both vasoconstrictors that inhibit its activity to cause MLC phosphorylation and contraction, and vasodilators that activate its activity to cause MLC dephosphorylation and relaxation. The RhoA/ROCK pathway is activated by vasoconstrictors to inhibit myosin phosphatase activity. The mechanism by which RhoA and ROCK are localized to and interact with myosin light chain phosphatase (MLCP) is not well understood. We recently found a new member of the myosin phosphatase complex, myosin phosphatase-rho interacting protein, that directly binds to both RhoA and the myosin-binding subunit of myosin phosphatase in vitro, and targets myosin phosphatase to the actinomyosin contractile filament in smooth muscle cells. Because myosin phosphatase-rho interacting protein binds both RhoA and MLCP, we investigated whether myosin phosphatase-rho interacting protein was required for RhoA/ROCK-mediated myosin phosphatase regulation. Myosin phosphatase-rho interacting protein silencing prevented LPA-mediated myosin-binding subunit phosphorylation, and inhibition of myosin phosphatase activity. Myosin phosphatase-rho interacting protein did not regulate the activation of RhoA or ROCK in vascular smooth muscle cells. Silencing of M-RIP lead to loss of stress fiber-associated RhoA, suggesting that myosin phosphatase-rho interacting protein is a scaffold linking RhoA to regulate myosin phosphatase at the stress fiber.     

RhoA and resistance artery remodeling

Resistance arteries are able to adapt to physiological and pathophysiological stimuli to maintain adequate perfusion according to the metabolic demand of the tissue. Although vasomotor control allows rapid adaptation of lumen diameter, vascular remodeling constitutes an active process that occurs in response to long-term alterations of hemodynamic parameters. Unfortunately, this initially adaptive process contributes to the pathology of vascular diseases. Recent studies have demonstrated the participation of Rho protein signaling pathways in several cardiovascular pathologies including hypertension, coronary artery spasm, effort angina, atherosclerosis, and restenosis. Functional analyses have further revealed that RhoA-dependent pathways are involved in excessive contraction, migration, and proliferation associated with arterial diseases. The present review focuses on the role of Rho proteins, in particular RhoA, in vascular smooth muscle cells and the involvement of Rho-dependent signaling pathways in resistance artery remodeling, more particularly in relation to hypertension.     

RhoGDI facilitates geranylgeranyltransferase-I-mediated RhoA prenylation

Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This modification is essential for the activity of small cellular GTPases, as it allows them to associate with intracellular membranes. Dissociated from membranes, prenylated proteins need to be transported through the aqueous cytoplasm by protein carriers that shield the hydrophobic anchor from the solvent. One such carrier is Rho GDP dissociation inhibitor (RhoGDI). Recently, it was shown that prenylated Rho proteins that are not associated with RhoGDI are subjected to proteolysis in the cell. We hypothesized that the role of RhoGDI might be not only to associate with prenylated proteins but also to regulate the prenylation process in the cell. This idea is supported by the fact that RhoGDI binds both unprenylated and prenylated Rho proteins with high affinity in vitro, and hence, these interactions may affect the kinetics of prenylation. We addressed this question experimentally and found that RhoGDI increased the catalytic efficiency of geranylgeranyl transferase-I in RhoA prenylation. Nevertheless, we did not observe formation of a ternary RhoGDI∗RhoA∗GGTase-I complex, indicating sequential operation of geranylgeranyltransferase-I and RhoGDI. Our results suggest that RhoGDI accelerates Rho prenylation by kinetically trapping the reaction product, thereby increasing the rate of product release.     

Thrombin-induced rapid geranylgeranylation of RhoA as an essential process for RhoA activation in endothelial cells

RhoA plays a critical signaling role in thrombin-induced endothelial dysfunction. The possible thrombin regulation of geranylgeranylation, a lipid modification, of unprocessed RhoA and the significance of the geranylgeranylation in RhoA activation in endothelial cells (ECs) are not well understood. The amounts of the unprocessed and geranylgeranylated forms of RhoA in non-stimulated cultured human aortic ECs were 31 +/- 8 and 69 +/- 8% total cellular RhoA, respectively (n = 6, p < 0.0001), as determined by the Triton X-114 partition method. Thrombin-induced rapid conversion of most of the unprocessed RhoA into the geranylgeranylated form within 1 min through stimulating geranylgeranyltransferase I (GGTase I) activity. Thrombin-induced rapid geranylgeranylation was inhibited by acute short term (3 min) pretreatment with atorvastatin as well as by an inhibitor of GGTase I (GGTI-286). Thrombin also rapidly stimulated GTP loading of RhoA, which was blocked by acute pretreatment with either atorvastatin or GGTI-286. These observations indicate the dependence of thrombin stimulation of RhoA on the rapid geranylgeranylation of unprocessed RhoA. Importantly, the addition of geranylgeranylpyrophosphate to ECs pretreated with atorvastatin quickly reversed the atorvastatin inhibition of thrombin stimulation of RhoA. These results suggest that geranylgeranylation of unprocessed RhoA may limit thrombin-induced full activation of RhoA in ECs. Cytoskeleton analysis demonstrated that atorvastatin and GGTI-286 inhibited thrombin-induced stress fiber formation. We provide the evidence that, in thrombin-stimulated ECs, the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which then is converted into GTP-bound active RhoA.     

Nicorandil regulates the macrophage skewing and ameliorates myofibroblasts by inhibition of RhoA/Rho‐kinase signalling in infarcted rats

We have demonstrated that ATP‐sensitive potassium (K_ATP) channel agonists attenuated fibrosis; however, the mechanism remained unclear. Since RhoA has been identified as a mediator of cardiac fibrosis, we sought to determine whether the anti‐fibrotic effects of K_ATP channel agonists were mediated via regulating macrophage phenotype and fibroblast differentiation by a RhoA/RhoA‐kinase‐dependent pathway. Wistar male rats after induction of myocardial infarction were randomized to either vehicle, nicorandil, an antagonist of K_ATP channel glibenclamide, an antagonist of ROCK fasudil, or a combination of nicorandil and glibenclamide or fasudil and glibenclamide starting 24 hrs after infarction. There were similar infarct sizes among the infarcted groups. At day 3 after infarction, post‐infarction was associated with increased RhoA/ROCK activation, which can be inhibited by administering nicorandil. Nicorandil significantly increased myocardial IL‐10 levels and the percentage of regulatory M2 macrophages assessed by immunohistochemical staining, Western blot, and RT‐PCR compared with vehicle. An IL‐10 receptor antibody increased myofibroblast infiltration compared with nicorandil alone. At day 28 after infarction, nicorandil was associated with attenuated cardiac fibrosis. These effects of nicorandil were functionally translated in improved echocardiographically derived cardiac performance. Fasudil showed similarly increased expression of M2 macrophages as nicorandil. The beneficial effects of nicorandil on fibroblast differentiation were blocked by adding glibenclamide. However, glibenclamide cannot abolish the attenuated fibrosis of fasudil, implying that RhoA/RhoA‐kinase is a downstream effector of K_ATP channel activation. Nicorandil polarized macrophages into M2 phenotype by inhibiting RhoA/RhoA‐kinase pathway, which leads to attenuated myofibroblast‐induced cardiac fibrosis after myocardial infarction.     

Phosphorylation-mediated regulation of GEFs for RhoA

Spatio-temporal control of RhoA GTPase is critical for regulation of cell migration, attachment to extracellular matrix, and cell–cell adhesions. Activation of RhoA is mediated by guanine nucleotide exchange factors (GEFs), a diverse family of enzymes that are controlled by multiple signaling pathways regulating actin cytoskeleton and cell migration. GEFs can be regulated by different mechanisms. Growing evidence demonstrates that phosphorylation serves as one of the predominant signals controlling activity, interactions, and localization of RhoGEFs. It acts as a positive and a negative regulator, and allows for regulation of RhoGEFs by multiple signaling cascades. Although there are common trends in phosphorylation-mediated regulation of some RhoGEF homologs, the majority of GEFs utilize distinct mechanisms that are dictated by their unique structure and interaction networks. This diversity enables multiple signaling pathways to use different RhoGEFs for regulation of a single central—RhoA. Here, we review current examples of phosphorylation-mediated regulation of GEFs for RhoA and its role in cell migration, discuss mechanisms, and provide insights into potential future directions.     

RhoA signaling in phorbol ester-induced apoptosis

Summary Exposure of cells to phorbol ester activates protein kinase C (PKC) to induce apoptosis or differentiation, depending on the cellular context. In erythroblastic cell lines, TF-1 and D2, upregulation of the RhoA signaling promotes phorbol ester-induced apoptosis through activating Rho-associated kinase (ROCK)/phosphorylation of myosin light chain (MLC), thus generating membrane contraction force. As a result, cell adhesion is inhibited and death receptor-mediated death pathway is activated in these cells with a concurrent changes in nucleocytoplasmic signaling for protein trafficking. A microtubule-regulated GEF-H1, which is a specific RhoA activator, was identified to contribute to RhoA activation in these cells. Thus, a cytoskeleton-regulated RhoA signaling cooperates with PKC activation constitutes a cellular context to determine the cell fate in response to phorbol ester stimulation.     

Modulating RhoA effectors induces transitions to oscillatory and more wavelike RhoA dynamics in Caenorhabditis elegans zygotes

Pulsatile RhoA dynamics underlie a wide range of cell and tissue behaviors. The circuits that produce these dynamics in different cells share common architectures based on fast positive and delayed negative feedback through F-actin, but they can produce very different spatiotemporal patterns of RhoA activity. However, the underlying causes of this variation remain poorly understood. Here we asked how this variation could arise through modulation of actin network dynamics downstream of active RhoA in early Caenorhabditis elegans embryos. We find that perturbing two RhoA effectors—formin and anillin—induce transitions from nonrecurrent focal pulses to either large noisy oscillatory pulses (formin depletion) or noisy oscillatory waves (anillin depletion). In both cases these transitions could be explained by changes in local F-actin levels and depletion dynamics, leading to changes in spatial and temporal patterns of RhoA inhibition. However, the underlying mechanisms for F-actin depletion are distinct, with different dependencies on myosin II activity. Thus, modulating actomyosin network dynamics could shape the spatiotemporal dynamics of RhoA activity for different physiological or morphogenetic functions.     

小G蛋白RhoA与细胞发育

细胞发育是细胞内各种复杂反应在时间和空间上有序协调的过程, 包括细胞增殖、胞质分裂、细胞运动、细胞分化和组织生成等．作为G蛋白家族重要成员的RhoA起了重要的调控作用：在G蛋白调控因子(如GEF XPLN、 p115RhoGEF、p190RhoGAP等）的作用下,活化的RhoA依次与效应蛋白分子（如ROCK1/2、 mDia、 PRK1/2、 citron 激酶等）结合, 从而开启了下游的信号通路, 最终使细胞能够迅速地对外界刺激做出反应．干细胞是一类既能自我更新又能特异分化形成终末分化细胞的细胞, 而 RhoA对干细胞的自我更新和定向分化也起着“开关"作用, 对RhoA信号通路的调节调控了胚胎发生、神经发生、造血生成及成骨和肌肉生成等干细胞分化发育过程．肿瘤是正常细胞在各种因素长期作用下增殖异常的产物, 而RhoA异常表达与肿瘤的发生、侵润与转移密切相关, RhoA信号通路与p53等基因的交互作用在肿瘤的发育过程中也发挥了重要的作用．     

Novel peptide recognized by RhoA GTPase

A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.     

RhoA inactivation enhances endothelial barrier function

The modulation of endothelial barrier function is thought to bea function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested this hypothesis by dissociating SF/FA withClostridium botulinum exoenzyme C3transferase (C3), an inhibitor of the small GTP-binding protein RhoA.Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using asize-selective permeability assay and quantitative digital imagingmeasurements of SF/FA. C3 treatment disassembled SF/FA, stimulateddiffuse myosin II immunostaining, and reduced the phosphotyrosine (PY)content of paxillin and 130- to 140-kDa proteins that includedp125^FAK. C3-treated monolayersdisplayed a 60-85% decline in F-actin content and a170-300% increase in EC surface area with enhanced endothelialbarrier function. This activity correlated with reorganization ofF-actin and PY protein(s) to -catenin-containing cell-cell junctions. Because C3 prevented the thrombin-induced formation ofmyosin ribbons, SF/FA, and the increased PY content of proteins, thesecharacteristics were Rho dependent. Our data show that C3 inhibition ofRho proteins leads to cAMP-like characteristics of reduced SF/FA andenhanced endothelial barrier function.