Differences in Gα12- and Gα13-mediated plasma membrane recruitment of p115-RhoGEF

Regulator of G protein signaling domain-containing Rho guanine-nucleotide exchange factors (RGS-RhoGEFs) directly links activated forms of the G12 family of heterotrimeric G protein α subunits to the small GTPase Rho. Stimulation of G_12/13-coupled GPCRs or expression of constitutively activated forms of α₁₂ and α₁₃ has been shown to induce the translocation of the RGS-RhoGEF, p115-RhoGEF, from the cytoplasm to the plasma membrane (PM). However, little is known regarding the functional importance and mechanisms of this regulated PM recruitment, and thus PM recruitment of p115-RhoGEF is the focus of this report. A constitutively PM-localized mutant of p115-RhoGEF shows a much greater activity compared to wild type p115-RhoGEF in promoting Rho-dependent neurite retraction of NGF-differentiated PC12 cells, providing the first evidence that PM localization can activate p115-RhoGEF signaling. Next, we uncovered the unexpected finding that Rho is required for α₁₃-induced PM translocation of p115-RhoGEF. However, inhibition of Rho did not prevent α₁₂-induced PM translocation of p115-RhoGEF. Additional differences between α₁₃ and α₁₂ in promoting PM recruitment of p115-RhoGEF were revealed by analyzing RGS domain mutants of p115-RhoGEF. Activated α₁₂ effectively recruits the isolated RGS domain of p115-RhoGEF to the PM, whereas α₁₃ only weakly does. On the other hand, α₁₃ strongly recruits to the PM a p115-RhoGEF mutant containing amino acid substitutions in an acidic region at the N-terminus of the RGS domain; however, α₁₂ is unable to recruit this p115-RhoGEF mutant to the PM. These studies provide new insight into the function and mechanisms of α_12/13-mediated PM recruitment of p115-RhoGEF.     

Redox Regulation of Ephrin/Integrin Cross-Talk

Interactions linking the Eph receptor tyrosine kinase and ephrin ligands transduce short-range repulsive signals regulating several motile biological processes including axon path-finding, angiogenesis and tumor growth. These ephrin-induced effects are believed to be mediated by alterations in actin dynamics and cytoskeleton reorganization. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. In particular, some ephrin ligands lead to a decrease in integrin-mediated cell adhesion and spread. Here we show that the ability of ephrinA1 to inhibit cell adhesion and spreading in prostatic carcinoma cells is strictly dependent on the decrease in the activity of the small GTPase Rac1. Given the recognized role of Rac-driven redox signaling for integrin function, reported to play an essential role in focal adhesion formation and in the overall organization of actin cytoskeleton, we investigated the possible involvement of oxidants in ephrinA1/EphA2 signaling. We now provide evidence that Reactive Oxygen Species are an integration point of the ephrinA1/integrin interplay. We identify redox circuitry in which the ephrinA1-mediated inhibition of Rac1 leads to a negative regulation of integrin redox signaling affecting the activity of the tyrosine phosphatase LMW-PTP. The enzyme in turn actively dephosphorylates its substrate p190RhoGAP, finally leading to RhoA activation. Altogether our data suggest a redox-based Rac-dependent upregulation of Rho activity, concurring with the inhibitory effect elicited by ephrinA1 on integrin-mediated adhesion strength.Key Words: EphA2 kinase, reactive oxygen species, integrin, cell repulsion, tumorigenesis     

Distinct Role of Pyk2 in Mediating Thromboxane Generation Downstream of Both G12/13 and Integrin αIIbβ3 in Platelets

Proline-rich tyrosine kinase 2 (Pyk2) is activated by various agonists in platelets. We evaluated the signaling mechanism and the functional role of Pyk2 in platelets by using pharmacological inhibitors and Pyk2-deficient platelets. We found that platelet aggregation and secretion in response to 2-methylthio-ADP (2-MeSADP) and AYPGKF were diminished in the presence of Pyk2 inhibitors or in Pyk2-deficient platelets, suggesting that Pyk2 plays a positive regulatory role in platelet functional responses. It has been shown that ADP-, but not thrombin-induced thromboxane (TxA₂) generation depends on integrin signaling. Unlike ADP, thrombin activates G_12/13 pathways, and G_12/13 pathways can substitute for integrin signaling for TxA₂ generation. We found that Pyk2 was activated downstream of both G_12/13 and integrin-mediated pathways, and both 2-MeSADP- and AYPGKF-induced TxA₂ generation was significantly diminished in Pyk2-deficient platelets. In addition, TxA₂ generation induced by co-stimulation of G_i and G_z pathways, which is dependent on integrin signaling, was inhibited by blocking Pyk2. Furthermore, inhibition of 2-MeSADP-induced TxA₂ generation by fibrinogen receptor antagonist was not rescued by co-stimulation of G_12/13 pathways in the presence of Pyk2 inhibitor. We conclude that Pyk2 is a common signaling effector downstream of both G_12/13 and integrin αIIbβ3 signaling, which contributes to thromboxane generation.     

An expression screen for RhoGEF genes involved in C. elegans gonadogenesis

The gonad in Caenorhabditis elegans is an important model system for understanding complex morphogenetic processes including cellular movement, cell fusion, cell invasion and cell polarity during development. One class of signaling proteins known to be critical for the cellular events underlying morphogenesis is the Rho family GTPases, particularly RhoA, Rac and Cdc42. In C. elegans orthologues of these genes have been shown to be important for gonad development. In our current study we have extended those findings by examining the patterns of 5′ cis-regulatory element (5′CRE) activity associated with nineteen putative guanine nucleotide exchange factors (GEFs) encoded by the C. elegans genome predicted to activate Rho family GTPases. Here we identify 13 RhoGEF genes that are expressed during gonadogenesis and characterize the cells in which their 5′CREs are active. These data provide the basis for designing experiments to examine Rho GTPase activation during morphogenetic processes central to normal gonad development.     

Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways

Microglia are major immunocompetent cells in the central nervous system and retain highly dynamic motility. The processes which allow these cells to move, such as chemotaxis and phagocytosis, are considered part of their functions and are closely related to purinergic signaling. Previously, we reported that the activation of the P2Y₆ receptor by UDP stimulation in microglia evoked dynamic cell motility which enhanced their phagocytic capacity, as reported by Koizumi et al. (Nature 446(7139):1091–1095, 2007). These responses require actin cytoskeletal rearrangement, which is seen after UDP stimulation. However, the intracellular signaling pathway has not been defined. In this study, we found that UDP in rat primary microglia rapidly induced the transient phosphorylation at Ser157 of vasodilator-stimulated phosphoprotein (VASP). VASP, one of actin binding protein, accumulated at the plasma membrane where filamentous (F)-actin aggregated in a time-dependent manner. The phosphorylation of VASP was suppressed by inhibition of PKC. UDP-induced local actin aggregations were also abrogated by PKC inhibitors. The Rho inhibitor CT04 and the expression of p115-RGS, which suppresses G_12/13 signaling, attenuated UDP-induced phosphorylation of VASP and actin aggregation. These results indicate that PKC- and Rho-dependent phosphorylation of VASP is involved in UDP-induced actin aggregation of microglia.     

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis,motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA₁, LPA₂, and LPA₃ on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.     

S1P(2) receptors mediate inhibition of glioma cell migration through Rho signaling pathways independent of PTEN

Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P₂ receptor, a carboxyl-terminal region of Gα12 or Gα13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P₂ receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P₂ receptors/G_12/13-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P₂ receptor-mediated action in glioma cells.     

LGR5 interacts and cointernalizes with Wnt receptors to modulate Wnt/β-catenin signaling

LGR5, a seven-transmembrane domain receptor of the rhodopsin family, is a Wnt target gene and a bona fide marker of adult stem cells in the gastrointestinal tract and hair follicle bulge. Recently, we and others demonstrated that LGR5 and its homologues function as receptors of the R-spondin family of stem cell factors to potentiate Wnt/β-catenin signaling. However, the mechanism of how LGR5 enhances the signaling output remains unclear. Here we report that following costimulation with the ligands R-spondin1 and Wnt3a, LGR5 interacts and forms a supercomplex with the Wnt coreceptors LRP6 and Fzd5 which is rapidly internalized and then degraded. Internalization of LGR5 is mediated through a dynamin- and clathrin-dependent pathway. Inhibition of this endocytic process has no effect on LGR5 signaling. Deletion of the C-terminal tail of LGR5 maintains its ability to interact with LRP6, yet this LGR5 mutant exhibits increased signaling activity and a decreased rate of endocytosis in response to R-spondin1 compared to the wild-type receptor. This study provides direct evidence that LGR5 becomes part of the Wnt signaling complex at the membrane level to enhance Wnt/β-catenin signaling. However, internalization of LGR5 does not appear to be essential for potentiating the canonical Wnt signaling pathway.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior

Using biochemical assays to determine the activation state of Rho-like GTPases, we show that the guanine nucleotide exchange factor Tiam1 functions as a specific activator of Rac but not Cdc42 or Rho in NIH3T3 fibroblasts. Activation of Rac by Tiam1 induces an epithelial-like morphology with functional cadherin-based adhesions and inhibits migration of fibroblasts. This epithelial phenotype is characterized by Rac-mediated effects on Rho activity. Transient PDGF-induced as well as sustained Rac activation by Tiam1 or V12Rac downregulate Rho activity. We found that Cdc42 also downregulates Rho activity. Neither V14Rho or N19Rho affects Rac activity, suggesting unidirectional signaling from Rac towards Rho. Downregulation of Rho activity occurs independently of Rac- induced cytoskeletal changes and cell spreading. Moreover, Rac effector mutants that are defective in mediating cytoskeleton changes or Jun kinase activation both downregulate Rho activity, suggesting that neither of these Rac signaling pathways are involved in the regulation of Rho. Restoration of Rho activity in Tiam1-expressing cells by expression of V14Rho results in reversion of the epithelioid phenotype towards a migratory, fibroblastoid morphology. We conclude that Rac signaling is able to antagonize Rho activity directly at the GTPase level, and that the reciprocal balance between Rac and Rho activity determines cellular morphology and migratory behavior in NIH3T3 fibroblasts.     

Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase

Coordinated cell movements shape simple epithelia into functional tissues and organs during embryogenesis. Regulators and effectors of the small GTPase Rho have been shown to be essential for epithelial morphogenesis in cell culture; however, the mechanism by which Rho GTPase and its downstream effectors control coordinated movement of epithelia in a developing tissue or organ is largely unknown. Here, we show that Rho1 GTPase activity is required for the invagination of Drosophila embryonic salivary gland epithelia and for directed migration of the internalized gland. We demonstrate that the absence of zygotic function of Rho1 results in the selective loss of the apical proteins, Crumbs (Crb), Drosophila atypical PKC and Stardust during gland invagination and that this is partially due to reduced crb RNA levels and apical localization. In parallel to regulation of crb RNA and protein, Rho1 activity also signals through Rho-kinase (Rok) to induce apical constriction and cell shape change during invagination. After invagination, Rho-Rok signaling is required again for the coordinated contraction and dorsal migration of the proximal half of the gland. We also show that Rho1 activity is required for proper development of the circular visceral mesoderm upon which the gland migrates. Our genetic and live-imaging analyses provide novel evidence that the proximal gland cells play an essential and active role in salivary gland migration that propels the entire gland to turn and migrate posteriorly.     

Notch signaling regulates gastric antral LGR5 stem cell function

The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5⁺ antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5‐GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5⁺ stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi‐colored reporter demonstrated that Notch‐activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD‐induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper‐proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.     

The C. elegans PH domain protein CED-12 regulates cytoskeletal reorganization via a Rho/Rac GTPase signaling pathway.

The C. elegans gene ced-12 functions in the engulfment of apoptotic cells and in cell migration, acting in a signaling pathway with ced-2 Crkll, ced-5 DOCK180, and ced-10 Rac GTPase and acting upstream of ced-10 Rac. ced-12 encodes a protein with a pleckstrin homology (PH) domain and an SH3 binding motif, both of which are important for ced-12 function. CED-12 acts in engulfing cells for cell corpse engulfment and interacts physically with CED-5, which contains an SH3 domain. CED-12 has Drosophila and human counterparts. Expression of CED-12 and its counterparts in murine Swiss 3T3 fibroblasts induced Rho GTPase-dependent formation of actin filament bundles. We propose that through interactions with membranes and with a CED-2/CED-5 protein complex, CED-12 regulates Rho/Rac GTPase signaling and leads to cytoskeletal reorganization by an evolutionarily conserved mechanism.     

Peripheral nerve demyelination caused by a mutant Rho GTPase guanine nucleotide exchange factor, frabin/FGD4

GTPases of the Rho subfamily are widely involved in the myelination of the vertebrate nervous system. Rho GTPase activity is temporally and spatially regulated by a set of specific guanine nucleotide exchange factors (GEFs). Here, we report that disruption of frabin/FGD4, a GEF for the Rho GTPase cell-division cycle 42 (Cdc42), causes peripheral nerve demyelination in patients with autosomal recessive Charcot-Marie-Tooth (CMT) neuropathy. These data, together with the ability of frabin to induce Cdc42-mediated cell-shape changes in transfected Schwann cells, suggest that Rho GTPase signaling is essential for proper myelination of the peripheral nervous system.     

LGR5/R-Spo1/Wnt3a axis promotes stemness and aggressive phenotype in hepatoblast-like hepatocellular carcinoma cell lines

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with “hepatoblast-like” appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and β-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/β-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/β-catenin pathway.