Non-canonical Wnt signals regulate cytoskeletal remodeling in osteoclasts

Osteoclasts are multinucleated cells responsible for bone resorption. Osteoclasts adhere to the bone surface through integrins and polarize to form actin rings, which are formed by the assembly of podosomes. The area contained within actin rings (also called sealing zones) has an acidic pH, which causes dissolution of bone minerals including hydroxyapatite and the degradation of matrix proteins including type I collagen by the protease cathepsin K. Osteoclasts resorb bone matrices while moving on bone surfaces. Osteoclasts change their cell shapes and exhibit three modes for bone resorption: motile resorbing mode for digging trenches, static resorbing mode for digging pits, and motile non-resorbing mode. Therefore, the actin cytoskeleton is actively remodeled in osteoclasts. Recent studies have revealed that many molecules, such as Rac, Cdc42, Rho, and small GTPase regulators and effectors, are involved in actin cytoskeletal remodeling during the formation of actin rings and resorption cavities on bone slices. In this review, we introduce how these molecules and non-canonical Wnt signaling regulate the bone-resorbing activity of osteoclasts.     

Plexin structures are coming: opportunities for multilevel investigations of semaphorin guidance receptors, their cell signaling mechanisms, and functions

Plexin transmembrane receptors and their semaphorin ligands, as well as their co-receptors (Neuropilin, Integrin, VEGFR2, ErbB2, and Met kinase) are emerging as key regulatory proteins in a wide variety of developmental, regenerative, but also pathological processes. The diverse arenas of plexin function are surveyed, including roles in the nervous, cardiovascular, bone and skeletal, and immune systems. Such different settings require considerable specificity among the plexin and semaphorin family members which in turn are accompanied by a variety of cell signaling networks. Underlying the latter are the mechanistic details of the interactions and catalytic events at the molecular level. Very recently, dramatic progress has been made in solving the structures of plexins and of their complexes with associated proteins. This molecular level information is now suggesting detailed mechanisms for the function of both the extracellular as well as the intracellular plexin regions. Specifically, several groups have solved structures for extracellular domains for plexin-A2, -B1, and -C1, many in complex with semaphorin ligands. On the intracellular side, the role of small Rho GTPases has been of particular interest. These directly associate with plexin and stimulate a GTPase activating (GAP) function in the plexin catalytic domain to downregulate Ras GTPases. Structures for the Rho GTPase binding domains have been presented for several plexins, some with Rnd1 bound. The entire intracellular domain structure of plexin-A1, -A3, and -B1 have also been solved alone and in complex with Rac1. However, key aspects of the interplay between GTPases and plexins remain far from clear. The structural information is helping the plexin field to focus on key questions at the protein structural, cellular, as well as organism level that collaboratoria of investigations are likely to answer.     

Actin dynamics in the regulation of endothelial barrier functions and neutrophil recruitment during endotoxemia and sepsis

Sepsis is a leading cause of death worldwide. Increased vascular permeability is a major hallmark of sepsis. Dynamic alterations in actin fiber formation play an important role in the regulation of endothelial barrier functions and thus vascular permeability. Endothelial integrity requires a delicate balance between the formation of cortical actin filaments that maintain endothelial cell contact stability and the formation of actin stress fibers that generate pulling forces, and thus compromise endothelial cell contact stability. Current research has revealed multiple molecular pathways that regulate actin dynamics and endothelial barrier dysfunction during sepsis. These include intracellular signaling proteins of the small GTPases family (e.g., Rap1, RhoA and Rac1) as well as the molecules that are directly acting on the actomyosin cytoskeleton such as myosin light chain kinase and Rho kinases. Another hallmark of sepsis is an excessive recruitment of neutrophils that also involves changes in the actin cytoskeleton in both endothelial cells and neutrophils. This review focuses on the available evidence about molecules that control actin dynamics and regulate endothelial barrier functions and neutrophil recruitment. We also discuss treatment strategies using pharmaceutical enzyme inhibitors to target excessive vascular permeability and leukocyte recruitment in septic patients.     

Cofilin activity during insulin-like growth factor I-stimulated neuroblastoma cell motility

Insulin-like growth factor I (IGF-I) is a potent stimulator of neuroblastoma cell motility. Cell motility requires lamellipodium extension at the leading edge of the cell through organized actin polymerization, and IGF-I stimulates lamellipodial elaboration in human neuroblastoma cells. Rac is a Rho GTPase that stimulates lamellipodial formation via the regulation of actin polymerization. In this study, we show that IGF-I-stimulated phosphatidylinositol 3-kinase (PI-3K) activity promotes rac activation and subsequent activation of the down- stream effectors LIM kinase and cofilin. Overexpression of wild-type LIM kinase and wild-type Xenopus ADF/cofilin (XAC) suppresses IGF-I-stimulated motility in SH-SY5Y cells, while expression of dominant negative LIM kinase and constitutively active XAC increases SH-SY5Y motility in the absence of IGF-I stimulation. These results suggest that regulation by cofilin of actin depolymerization is important in the process of neuroblastoma cell motility, and IGF-I regulates cofilin activity in part through PI-3K, rac, and LIM kinase.Received 18 October 2004; received after revision 3 December 2004; accepted 16 December 2004     

Effects of constitutively active GTPases on fibroblast behavior

The GTP-binding proteins RhoA, Cdc42 and Rac1 regulate the organization and turnover of the cytoskeleton and cell-matrix adhesions, structures bridging cells to their support, and translating forces, external or generated within the cell. To investigate the specific requirements of Rho GTPases for biomechanical activities of clonal cell populations, we compared side-by-side stable lines of human fibroblasts expressing constitutively active (CA) RhoA, Cdc42 or Rac1. There was no marked effect of any CA GTPase on cell adhesion to different extracellular matrix proteins. Cell spreading was CA Rho GTPase specific and independent of the extracellular matrix proteins allowing adhesion. Mechanical properties were dramatically restricted by CA RhoA on bi- and in tri-dimensional surroundings, were boosted by CA Rac1 on bi-dimensional surroundings only, and were not or marginally affected by CA Cdc42. In conclusion, the action of Rho GTPases appears to depend on the task cells are performing. Received 12 September 2005; received after revision 5 October 2005; accepted 1 November 2005     

The parvins

The parvins are a family of proteins involved in linking integrins and associated proteins with intracellular pathways that regulate actin cytoskeletal dynamics and cell survival. Both α-parvin (PARVA) and β-parvin (PARVB) localize to focal adhesions and function in cell adhesion, spreading, motility and survival through interactions with partners, such as integrin-linked kinase (ILK), paxillin, α-actinin and testicular kinase 1. A complex of PARVA with ILK and the LIM protein PINCH-1 is critical for cell survival in a variety of cells, including certain cancer cells, kidney podocytes and cardiac myocytes. While PARVA inhibits the activities of Rac1 and testicular kinase 1 and cell spreading, PARVB binds αPIX and α-actinin, and can promote cell spreading. In contrast to PARVA, PARVB inhibits ILK activity and reverses some of its oncogenic effects in cancer cells. This review focuses on the structure and function of the parvins and some possible roles in human diseases. Received 5 August 2005; received after revision 5 September 2005; accepted 22 September 2005     

A matricellular protein and EGF-like repeat signalling in the social amoebozoan Dictyostelium discoideum

Matricellular proteins interact with the extracellular matrix (ECM) and modulate cellular processes by binding to cell surface receptors and initiating intracellular signal transduction. Their association with the ECM and the ability of some members of this protein family to regulate cell motility have opened up new avenues of research to investigate their functions in normal and diseased cells. In this review, we summarize the research on CyrA, an ECM calmodulin-binding protein in Dictyostelium. CyrA is proteolytically cleaved into smaller EGF-like (EGFL) repeat containing cleavage products during development. The first EGFL repeat of CyrA binds to the cell surface and activates a novel signalling pathway that modulates cell motility in this model organism. The similarity of CyrA to the most well-characterized matricellular proteins in mammals allows it to be designated as the first matricellular protein identified in Dictyostelium.     

Bacterial LPX motif-harboring virulence factors constitute a species-spanning family of cell-penetrating effectors

Effector proteins are key virulence factors of pathogenic bacteria that target and subvert the functions of essential host defense mechanisms. Typically, these proteins are delivered into infected host cells via the type III secretion system (T3SS). Recently, however, several effector proteins have been found to enter host cells in a T3SS-independent manner thereby widening the potential range of these virulence factors. Prototypes of such bacteria-derived cell-penetrating effectors (CPEs) are the Yersinia enterocolitica-derived YopM as well as the Salmonella typhimurium effector SspH1. Here, we investigated specifically the group of bacterial LPX effector proteins comprising the Shigella IpaH proteins, which constitute a subtype of the leucine-rich repeat protein family and share significant homologies in sequence and structure. With particular emphasis on the Shigella-effector IpaH9.8, uptake into eukaryotic cell lines was shown. Recombinant IpaH9.8 (rIpaH9.8) is internalized via endocytic mechanisms and follows the endo-lysosomal pathway before escaping into the cytosol. The N-terminal alpha-helical domain of IpaH9.8 was identified as the protein transduction domain required for its CPE ability as well as for being able to deliver other proteinaceous cargo. rIpaH9.8 is functional as an ubiquitin E3 ligase and targets NEMO for poly-ubiquitination upon cell penetration. Strikingly, we could also detect other recombinant LPX effector proteins from Shigella and Salmonella intracellularly when applied to eukaryotic cells. In this study, we provide further evidence for the general concept of T3SS-independent translocation by identifying novel cell-penetrating features of these LPX effectors revealing an abundant species-spanning family of CPE.     

ROPs in the spotlight of plant signal transduction

Small guanine nucleotide binding proteins of the Rho family called ROP play a crucial role as regulators of signal transduction in plants. They participate in pathways that influence growth and development, and the adaptation of plants to various environmental situations. As members of the Ras superfamily, ROPs function as molecular switches cycling between a GDP-bound ‘off’ and a GTP-bound ‘on’ state in a strictly regulated manner. Latest research provided fascinating new insights into ROP regulation by novel guanine nucleotide exchange factors, unconventional GTPase activating proteins, and guanine nucleotide dissociation inhibitors, which apparently organize localized ROP activation. Important progress has also been made concerning signaling components upstream and downstream of the ROP cycle involving receptor-like serine/threonine kinases and effectors that can manipulate cytoskeletal dynamics, intracellular calcium levels, H₂O₂ production and further cellular targets. This review outlines the fast developing knowledge on ROP GTPases highlighting their specific features, regulation and roles in a cellular signaling context. Received 28 April 2006; received after revision 2 June 2006; accepted 29 June 2006     

Sphingosine 1-phosphate increases glucose uptake through trans-activation of insulin receptor

Sphingosine 1-phosphate (S1P) is a bioactive lipid that acts through a family of G-protein-coupled receptors. Herein, we report evidence of a novel redox-based cross-talk between S1P and insulin signaling pathways. In skeletal muscle cells S1P, through engagement of its S1P₂ receptor, is found to produce a transient burst of reactive oxygen species through a calcium-dependent activation of the small GTPase Rac1. S1P-induced redox-signaling is sensed by protein tyrosine phosphatase-1B, the main negative regulator of insulin receptor phosphorylation, which undergoes oxidation and enzymatic inhibition. This redox-based inhibition of the phosphatase provokes a ligand-independent trans-phosphorylation of insulin receptor and a strong increase in glucose uptake. Our results propose a new role of S1P, recognizing the lipid as an insulin-mimetic cue and pointing at reactive oxygen species as critical regulators of the cross-talk between S1P and insulin pathways. Any possible implication of S1P-directed insulin signaling in diabetes and insulin resistance remains to be established.     

Pressure activates colon cancer cell adhesion via paxillin phosphorylation, Crk, Cas, and Rac1

Physical forces can activate colon cancer cell adhesion, critical for metastasis. Paxillin is phosphorylated by FAK and required for pressure-stimulated adhesion. However, whether paxillin acts as an inert scaffolding protein or whether paxillin phosphorylation is required is unknown. Transfection with paxillin point-phosphorylation mutants demonstrated that phosphorylation at tyrosines 31 and 118 together is necessary for pressure-stimulated adhesion. We further evaluated potential paxillin partners. Reducing the adaptor protein Crk or the focal adhesion protein p130^Cas blocked pressure-stimulated adhesion. Furthermore, Crk and p130^Cas both displayed increased co-immunoprecipitation with paxillin in response to increased pressure, except in cells transfected with a Y31Y118 paxillin mutant. Inhibiting the small GTPase Rac1 also abolished pressure-stimulated adhesion, and reducing paxillin by siRNA blocked Rac1 phosphorylation by pressure. Thus, paxillin phosphorylation at tyrosines 31 and 118 together is necessary for pressure-induced adhesion. Paxillin, Crk and Cas form a trimeric complex that activates Rac1 and mediates this effect. Received 21 January 2008; received after revision 4 March 2008; accepted 19 March 2008     

Targeting of the Akt/PKB kinase to the actin skeleton

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.Received 10 September 2003; accepted 25 September 2003     

Kank proteins: structure, functions and diseases

The Kank family of proteins, Kank1–Kank4, are characterized by their unique structure, coiled-coil motifs in the N-terminal region, and ankyrin-repeats in the C-terminal region, with an additional motif, the KN motif, at the N-terminus. Kank1 was obtained by positional cloning of a tumor suppressor gene in renal cell carcinoma, while the other members were found by homology search. The family is involved in the regulation of actin polymerization and cell motility through signaling pathways containing PI3K/Akt and/or unidentified modulators/effectors. Their relationship to diseases such as cancer, and to neuronal and developmental disorders, will be an important subject of future study.     

Sequential inactivation of Rho GTPases and Lim kinase by Pseudomonas aeruginosa toxins ExoS and ExoT leads to endothelial monolayer breakdown

Pseudomonas aeruginosa is a major human opportunistic pathogen and one of the most important causal agents of bacteremia. For non-blood-borne infection, bacterial dissemination requires the crossing of the vascular endothelium, the main barrier between blood and the surrounding tissues. Here, we investigated the effects of P. aeruginosa type 3 secretion effectors, namely ExoS, ExoT, and ExoY, on regulators of actin cytoskeleton dynamics in primary endothelial cells. ExoS and ExoT similarly affected the Lim kinase-cofilin pathway, thereby promoting actin filament severing. Cofilin activation was also observed in a mouse model of P. aeruginosa-induced acute pneumonia. Rho, Rac, and Cdc42 GTPases were sequentially inactivated, leading to inhibition of membrane ruffling, filopodia, and stress fiber collapse, and focal adhesion disruption. At the end of the process, ExoS and ExoT produced a dramatic retraction in all primary endothelial cell types tested and thus a rupture of the endothelial monolayer. ExoY alone had no effect in this context. Cell retraction could be counteracted by overexpression of actin cytoskeleton regulators. In addition, our data suggest that moesin is neither a direct exotoxin target nor an important player in this process. We conclude that any action leading to inhibition of actin filament breakdown will improve the barrier function of the endothelium during P. aeruginosa infection.     

Overactive bone morphogenetic protein signaling in heterotopic ossification and Duchenne muscular dystrophy

Bone morphogenetic proteins (BMPs) are important extracellular cytokines that play critical roles in embryogenesis and tissue homeostasis. BMPs signal via transmembrane type I and type II serine/threonine kinase receptors and intracellular Smad effector proteins. BMP signaling is precisely regulated and perturbation of BMP signaling is connected to multiple diseases, including musculoskeletal diseases. In this review, we will summarize the recent progress in elucidation of BMP signal transduction, how overactive BMP signaling is involved in the pathogenesis of heterotopic ossification and Duchenne muscular dystrophy, and discuss possible therapeutic strategies for treatment of these diseases.     

Impaired Rho GTPase activation abrogates cell polarization and migration in macrophages with defective lipolysis

Infiltration of monocytes and macrophages into the site of inflammation is critical in the progression of inflammatory diseases such as atherosclerosis. Cell migration is dependent on the continuous organization of the actin cytoskeleton, which is regulated by members of the small Rho GTPase family (RhoA, Cdc42, Rac) that are also important for the regulation of signal transduction pathways. We have recently reported on reduced plaque formation in an atherosclerotic mouse model transplanted with bone marrow from adipose triglyceride lipase-deficient (Atgl-/-) mice. Here we provide evidence that defective lipolysis in macrophages lacking ATGL, the major enzyme responsible for triacylglycerol hydrolysis, favors an anti-inflammatory M2-like macrophage phenotype. Our data implicate an as yet unrecognized principle that insufficient lipolysis influences macrophage polarization and actin polymerization, resulting in impaired macrophage migration. Sustained phosphorylation of focal adhesion kinase [due to inactivation of its phosphatase by elevated levels of reactive oxygen species (ROS)] results in defective Cdc42, Rac1 and RhoA activation and in increased and sustained activation of Rac2. Inhibition of ROS production restores the migratory capacity of Atgl-/- macrophages. Since monocyte and macrophage migration are a prerequisite for infiltrating the arterial wall, our results provide a molecular link between lipolysis and the development of atherosclerosis.     

Decoding the Hedgehog signal in animal development

The Hedgehog (Hh) family of secreted proteins plays essential roles in a myriad of developmental processes via a complex signaling cascade conserved in species ranging from insects to mammals. In many developmental contexts, Hh acts as long-range morphogen to control distinct cellular outcomes as a function of its concentration. Here we review the current understanding of the Hh signaling mechanisms that govern the establishment of the Hh gradient and the transduction of the Hh signal with an emphasis on the intracellular signaling cascade from the receptor to the nuclear effector. We discuss how graded Hh signals are transduced to govern distinct developmental outcomes. Received 28 October 2005; received after revision 6 February 2006; accepted 15 February 2006     

PAR3–PAR6–atypical PKC polarity complex proteins in neuronal polarization

Polarity is a fundamental feature of cells. Protein complexes, including the PAR3–PAR6–aPKC complex, have conserved roles in establishing polarity across a number of eukaryotic cell types. In neurons, polarity is evident as distinct axonal versus dendritic domains. The PAR3, PAR6, and aPKC proteins also play important roles in neuronal polarization. During this process, either aPKC kinase activity, the assembly of the PAR3–PAR6–aPKC complex or the localization of these proteins is regulated downstream of a number of signaling pathways. In turn, the PAR3, PAR6, and aPKC proteins control various effector molecules to establish neuronal polarity. Herein, we discuss the many signaling mechanisms and effector functions that have been linked to PAR3, PAR6, and aPKC during the establishment of neuronal polarity.     

Akt-mediated GSK-3β inhibition prevents migration of polyamine-depleted intestinal epithelial cells via Rac1

The rapid migration of intestinal epithelial cells (IEC) is important for the healing of mucosal wounds. We have previously shown that polyamine depletion inhibits migration of IEC-6 cells. Akt activation and its downstream target GSK-3β have been implicated in the regulation of migration. Here we investigated the significance of elevated phosphatidylinositol 3-kinase (PI3K)/Akt signaling on migration of polyamine-depleted cells. Polyamine-depleted cells had high Akt (Ser473) and GSK-3β (Ser9) phosphorylation. Pretreatment with 20 μM LY294002 (PI3K inhibitor) for 30 min inhibited phosphorylation of Akt, increased migration by activating Rac1 in polyamine-depleted IEC-6 cells, and restored the actin structure similar to that in cells grown in control medium. Treatment of cells with a GSK-3β inhibitor (AR-A014418) altered the actin cytoskeleton and inhibited migration, mimicking the effects of polyamine depletion. Thus, our results indicate that sustained activation of Akt in response to polyamine depletion inhibits migration through GSK-3β and Rac1. Received 25 August 2006; received after revision 3 October 2006; accepted 16 October 2006