Rho GTPases are involved in S1P‐enhanced glomerular endothelial cells activation with anti‐myeloperoxidase antibody positive IgG

Sphingosine‐1‐phosphate (S1P) is a crucial regulator in vascular inflammation. Our recent study found that under pathophysiological concentration in active anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV), S1P participated in MPO‐ANCA‐positive IgG‐induced glomerular endothelial cell (GEnC) activation via a S1P receptor (S1PR)‐dependent way. However, the downstream signalling pathways are not fully clear yet. In this study, we demonstrated that Rho guanosine triphosphatases (GTPases) signalling pathways, RhoA and Rac1 in particular, were implicated in MPO‐ANCA‐positive IgG‐mediated GEnCs activation enhanced by pathophysiological concentration of S1P in AAV. These results provide mechanistic insights into vascular barrier dysfunction in AAV, which may facilitate the development of effective therapies.     

Lentiviral siRNA silencing of sphingosine-1-phosphate receptors S1P1 and S1P2 in smooth muscle

Sphingosine-1-phosphate regulates diverse biological processes through five receptor types, S1P(1-5). Two or more S1P receptors are usually co-expressed on target cells. We have previously shown that smooth muscle cells of the gut co-express S1P(1) and S1P(2) receptors that could mediate distinct functions. In the absence of selective agonists and antagonists, we developed siRNA constructs to silence each receptor separately. The constructs were based on identical sequences in several mammalian species. A lentiviral vector-based system was used to deliver siRNA into HEK293T cells and smooth muscle cells. One S1P(1) and two S1P(2) siRNA constructs specifically inhibited ectopic expression of S1P(1) and S1P(2) receptors, respectively, as determined by immunocytochemistry and Western blot, and endogenous expression of S1P(1) and S1P(2) receptors in smooth muscle cells, as determined by RT-PCR. Measurement of PLC-beta and Rho kinase activities, which mediate initial and sustained muscle contraction, confirmed receptor silencing and showed that contraction is mediated exclusively by S1P(2) receptors.     

Caveolin-1 in cell polarization and directional migration

Migration is a complex process in which cells move in a given direction either in response to changes in the extracellular environment or as a consequence of an intrinsic propensity for directional movement. Migration plays key roles in many physiological and pathological processes, including development, angiogenesis, tissue regeneration and metastasis. An important role in migration is played by caveolin-1 and caveolae. Caveolae compartmentalize intracellular signalling pathways to orchestrate cell migration. Caveolin-1 presents a polarized distribution in migrating cells and is linked to the cytoskeleton, and changes in its expression modulate migration. Although there are some discrepancies regarding the regulatory effect of caveolin-1, most studies show that it promotes cell movement and polarity. The importance of caveolin-1 has recently been reinforced by studies with Cav1(-/-) cells, which indicate that it establishes polarity during directional migration by coordinating Src kinase and Rho GTPase signalling.     

P21-activated kinase 4 – Not just one of the PAK

P21-activated kinase 4 (PAK4) is a member of the p21-activated kinase (PAK) family. Historically much of the attention has been directed towards founding family member PAK1 but the focus is now shifting towards PAK4. It is a pluripotent serine/threonine kinase traditionally recognised as a downstream effector of the Rho-family GTPases. However, emerging research over the last few years has revealed that this kinase is much more than that. New findings have shed light on the molecular mechanism of PAK4 activation and how this kinase is critical for early development. Moreover, the number of PAK4 substrates and binding partners is rapidly expanding highlighting the increasing amount of cellular functions controlled by PAK4. We propose that PAK4 should be considered a signalling integrator regulating numerous fundamental cellular processes, including actin cytoskeletal dynamics, cell morphology and motility, cell survival, embryonic development, immune defence and oncogenic transformation. This review will outline our current understanding of PAK4 biology.     

T-cadherin activates Rac1 and Cdc42 and changes endothelial permeability

In the present study, expression of T-cadherin was shown to induce intracellular signaling in NIH3T3 fibroblasts: it activated Rac1 and Cdc42 (p < 0.01) but not RhoA. T-Cadherin overexpression in human umbilical vein endothelial cells (HUVEC) using adenoviral constructs induced disassembly of microtubules and polymerization of actin stress fibers, whereas down-regulation of endogenous T-cadherin expression in HUVEC using lentiviral constructs resulted in micro-tubule polymerization and a decrease in the number of actin stress fibers. Moreover, suppression of the T-cadherin expression significantly decreased the endothelial monolayer permeability as compared to the control (p < 0.001).     

Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction

Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na(+) and Ca(2+) current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up-regulation of matrix metalloproteinase 9 (MMP-9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease.     

PAR1-mediated RhoA activation facilitates CCL2-induced chemotaxis in PC-3 cells

Patients with advanced prostate cancer often exhibit increased activation of the coagulation system. The key activator of the coagulation cascade is the serine protease thrombin which is capable of eliciting numerous cellular responses. We previously reported that the thrombin receptor PAR1 is overexpressed in prostate cancer. To investigate further the role of PAR1 in prostate cancer metastasis, we examined the effects of thrombin activation on cell adhesion and motility in PC-3 prostate cancer cells. Activation of PAR1-induced dynamic cytoskeletal reorganization and reduced PC-3 binding to collagen I, collagen IV, and laminin (P < 0.01) but not fibronectin. Expression of the cell surface integrin receptors did not change as assessed by flow cytometry. Immunofluorescence microscopy revealed that PAR1 stimulation caused reorganization of the focal adhesions, suggesting that PAR1 activation in PC-3 cells may be modulating cell adhesion through integrin function but not expression. Furthermore, RhoA was activated upon stimulation with thrombin with subsequent cell contraction, decreased cell adhesion, and induced migration towards monocyte chemoattractant protein 1 (MCP-1; CCL2). Thus, it appears that thrombin stimulation plays a role in prostate cancer metastasis by decreasing cell adhesion to the extracellular matrix and positioning the cell in a "ready state" for migration in response to a chemotactic signal. Further exploration is needed to determine whether PAR1 activation affects other signaling pathways involved in prostate cancer.     

RhoB affects macrophage adhesion, integrin expression and migration

Rho GTPases regulate multiple cellular responses, including cell motility and cell cycle progression. The Rho isoform RhoB represses transformation and affects endosomal trafficking, but its effects on cell adhesion and migration have not been investigated in detail. Here we show that RhoB-null macrophages are more rounded than wild-type macrophages on fibronectin and uncoated glass, and have reduced adhesion to ICAM-1 and glass but not fibronectin. This correlated with lower cell surface expression of beta2 and beta3 integrins but not beta1 integrin. RhoB-null cells migrated faster than Wt cells on fibronectin, consistent with their smaller spread area, but slower than Wt cells on glass, reflecting their reduced adhesion. C3 transferase, which inhibits RhoA, RhoB and RhoC, induced cell spreading but this effect was reduced in RhoB-null cells. However, RhoB is not required for assembly of podosomes, which are integrin-based adhesion sites, whereas C3 transferase induced a decrease in podosomes and defects in tail retraction. Since macrophages do not express RhoC, these effects of C3 transferase are due to inhibition of RhoA rather than RhoB. Our results suggest that RhoB affects cell shape and migration by regulating surface integrin levels.     

Rho GTPases与骨髓间充质干细胞的迁移和分化

Rho家族小分子鸟苷三磷酸酶（small GTPases of Rho family,Rho GTPases）是调节细胞许多生理病理活动的关键分子开关,参与细胞骨架、基因转录、细胞周期进程、细胞黏附的调控及多条信号通路的调节。骨髓间充质干细胞（bone marrow-derived mesenchymal stem cells,MSCs）是一类具有自我更新和多向分化潜能的特殊细胞,通过增殖、迁移、分化等途径参与机体损伤组织的修复过程。研究表明,Rho GT-Pases在MSCs迁移、分化等过程中起着重要的调节作用。     

Glycogen synthase kinase-3 regulates cytoskeleton and translocation of Rac1 in long cellular extensions of human keratinocytes

Wound keratinocytes form long cellular extensions that facilitate their migration from the wound edge into provisional matrix. We have previously shown that similar extensions can be induced by a long-term exposure to EGF or rapidly by staurosporine in cultured cells. This morphological change depends on the activity of glycogen synthase kinase-3 (GSK-3). Here, we have characterized the cytoskeletal changes involved in formation of these extended lamellipodia (E-lam) in human HaCaT keratinocytes. E-lams contained actin filaments, stable microtubules and keratin intermediate filaments. E-lam formation was prevented by cytochalasin D, colchicine and low concentrations of taxol and nocodazole, suggesting that actin and microtubule organization and dynamics are essential for E-lam formation. Staurosporine induced recruitment of filamentous actin (F-actin), cortactin, filamin, Arp2/3 complex, Rac1 GTPase and phospholipase C-gamma1 (PLC-gamma1) to lamellipodia. Treatment of cells with the GSK-3 inhibitors SB-415286 and LiCl(2) inhibited E-lam formation and prevented the accumulation of Rac1 and Arp2/3 complex at lamellipodia. The formation of E-lams was dependent on fibronectin-binding integrins and normally regulated Rac1, and expression of either dominant-negative or constitutively active forms of Rac1 prevented E-lam formation. Overexpression of either RhoA or Cdc42 GTPases suppressed E-lam formation. We conclude that extended lamellipodia formation in keratinocytes requires actin and tubulin assembly at the leading edge, and this process is regulated by Rac1 downstream of GSK-3.     

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.     

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.     

Roles of Rho GTPases in leucocyte and leukaemia cell transendothelial migration

Leucocytes migrate into and out of blood vessels at multiple points during their development and maturation, and during immune surveillance. In response to tissue damage and infection, they are rapidly recruited through the endothelium lining blood vessels into the tissues. Leukaemia cells also move in and out of the bloodstream during leukaemia progression. Rho GTPases are intracellular signalling proteins that regulate cytoskeletal dynamics and are key coordinators of cell migration. Here, we describe how different members of the Rho GTPase family act in leucocytes and leukaemia cells to regulate steps of transendothelial migration. We discuss how inhibitors of Rho signalling could be used to reduce leucocyte or leukaemia cell entry into tissues.     

Traction forces of fibroblasts are regulated by the Rho-dependent kinase but not by the myosin light chain kinase

Adhesive cells show complex mechanical interactions with the substrate, however the exact mechanism of such interactions, termed traction forces, is still unclear. To address this question we have measured traction forces of fibroblasts treated with agents that affect the myosin II-dependent contractile mechanism. Using the potent myosin II inhibitor blebbistatin, we demonstrate that traction forces are strongly dependent on a functional myosin II heavy chain. Since myosin II is regulated by both the myosin light chain kinase (MLCK) and, directly or indirectly, the Rho-associated kinase (ROCK), we examined the effects of inhibitors against these kinases. Interestingly, inhibition of the myosin light chain kinase had no detectable effect, while inhibition of the Rho-dependent kinase caused strong inhibition of traction forces. Our results indicate that ROCK and MLCK play non-redundant roles in regulating myosin II functions, and that a subset of myosin II, regulated by the Rho small GTPase, may be responsible for the regulation of traction forces in migrating fibroblasts.     

Membrane trafficking at the ER/Golgi interface: functional implications of RhoA and Rac1

N-WASP and Arp2/3, the components of the actin nucleation/polymerization signaling pathway governed by Cdc42, are located in Golgi membranes and regulate ER/Golgi interface protein transport. In the present study, we examined whether RhoA and Rac1, like Cdc42, are also involved in this early secretory pathway. Unlike Cdc42, RhoA and Rac1 were not observed in the Golgi complex of different clonal cell lines nor were they present in isolated Golgi membranes. Expression of constitutively active or inactive mutants of RhoA or Rac1 proteins in HeLa cells did not alter either the disassembly or the assembly of the Golgi complex following the addition or withdrawal of BFA, respectively, the ER-to-Golgi VSV-G transport or the Sar1(dn)-induced ER accumulation of Golgi proteins. Moreover, unlike Cdc42-expressing cells, the 15 degrees C-induced subcellular redistribution of the KDEL receptor remained unaltered. Only cells that constitutively express the activated Cdc42 mutant (Cdc42Q61L), or that were microinjected with activated Cdc42Q61L protein, exhibited a significant change in Golgi complex morphology. Collectively, our results demonstrate that RhoA and Rac1 are not located in the Golgi complex, nor do they directly or indirectly regulate membrane trafficking at the ER/Golgi interface. This finding, in turn, confirms that Cdc42 is the only Rho GTPase to have a specific function on the Golgi complex.     

Shaping the landscape: Metabolic regulation of S1P gradients

Sphingosine-1-phosphate (S1P) is a lipid that functions as a metabolic intermediate and a cellular signaling molecule. These roles are integrated when compartments with differing extracellular S1P concentrations are formed that serve to regulate functions within the immune and vascular systems, as well as during pathologic conditions. Gradients of S1P concentration are achieved by the organization of cells with specialized expression of S1P metabolic pathways within tissues. S1P concentration gradients underpin the ability of S1P signaling to regulate in vivo physiology. This review will discuss the mechanisms that are necessary for the formation and maintenance of S1P gradients, with the aim of understanding how a simple lipid controls complex physiology. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Individual variation of human S1P1 coding sequence leads to heterogeneity in receptor function and drug interactions

Sphingosine 1-phosphate receptor 1 (S1P₁), an abundantly-expressed G protein-coupled receptor which regulates key vascular and immune responses, is a therapeutic target in autoimmune diseases. Fingolimod/Gilenya (FTY720), an oral medication for relapsing-remitting multiple sclerosis, targets S1P₁ receptors on immune and neural cells to suppress neuroinflammation. However, suppression of endothelial S1P₁ receptors is associated with cardiac and vascular adverse effects. Here we report the genetic variations of the S1P₁ coding region from exon sequencing of >12,000 individuals and their functional consequences. We conducted functional analyses of 14 nonsynonymous single nucleotide polymorphisms (SNPs) of the S1PR1 gene. One SNP mutant (Arg¹²⁰ to Pro) failed to transmit sphingosine 1-phosphate (S1P)-induced intracellular signals such as calcium increase and activation of p44/42 MAPK and Akt. Two other mutants (Ile⁴⁵ to Thr and Gly³⁰⁵ to Cys) showed normal intracellular signals but impaired S1P-induced endocytosis, which made the receptor resistant to FTY720-induced degradation. Another SNP mutant (Arg¹³ to Gly) demonstrated protection from coronary artery disease in a high cardiovascular risk population. Individuals with this mutation showed a significantly lower percentage of multi-vessel coronary obstruction in a risk factor-matched case-control study. This study suggests that individual genetic variations of S1P₁ can influence receptor function and, therefore, infer differential disease risks and interaction with S1P₁-targeted therapeutics.     

鞘氨醇激酶1和1-磷酸鞘氨醇受体2在癫痫大鼠海马组织中表达的变化*

目的:检测鞘氨醇激酶1 (SphK1)和1-磷酸鞘氨醇受体2 (S1PR2) 在癫痫大鼠海马中的表达,探讨SphK1和S1PR2在癫痫中的作用机制。方法:成年雄性SD大鼠108只,随机分为对照(Control)组(n=48)和癫痫(PILO)组(n=60)。癫痫组腹腔注射氯化锂(127 mg/kg),18～20 h后注射匹罗卡品,首剂量为30 mg/kg,发作＜IV级的大鼠重复注射匹罗卡品(10 mg/kg);对照组给予等剂量的生理盐水代替匹罗卡品。根据造模后观察时间和行为学改变,随机分为3个大组,6个亚组:急性期组(E6 h、E1 d、E3 d)、潜伏期组(E7 d)和慢性期组(E30 d、E56 d),每个亚组中对照大鼠和癫痫大鼠各8只。每组取4只大鼠麻醉取海马,另4只取大脑组织。运用Western blot检测SphK1、S1PR2在大鼠海马组织中的表达变化,免疫荧光检测星形胶质细胞活化增生情况及SphK1、S1PR2在星形胶质细胞中的定位表达。结果:与Control组比较,SphK1在造模后急性期(E3 d)、潜伏期(E7 d)和慢性期(E30 d、E56 d)海马中的表达均明显升高(P＜0.05或P＜0.01);S1PR2在急性期(E3 d)、潜伏期(E7 d)和慢性期(E30 d、E56 d)海马组织中的表达均明显下降(P＜0.05或P＜0.01);癫痫大鼠(E7 d)海马星形胶质细胞活化、增生明显(P＜0.05),SphK1和S1PR2在E7d的表达到位为海马星形胶质细胞中。结论:SphK1和S1PR2可能通过调控海马星形胶质细胞活化增生和影响神经元兴奋性参与了癫痫的发病。     

Platelet-derived growth factors and their receptors: Structural and functional perspectives

The four types of platelet-derived growth factors (PDGFs) and the two types of PDGF receptors (PDGFRs, which belong to class III receptor tyrosine kinases) have important functions in the development of connective tissue cells. Recent structural studies have revealed novel mechanisms of PDGFs in propeptide loading and receptor recognition/activation. The detailed structural understanding of PDGF–PDGFR signaling has provided a template that can aid therapeutic intervention to counteract the aberrant signaling of this normally silent pathway, especially in proliferative diseases such as cancer. This review summarizes the advances in the PDGF system with a focus on relating the structural and functional understandings, and discusses the basic aspects of PDGFs and PDGFRs, the mechanisms of activation, and the insights into the therapeutic antagonism of PDGFRs. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.