C3 Botulinum Toxin Research Articles

A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

Rho Family Guanine Nucleotide Exchange Factor Brx Couples Extracellular Signals to the Glucocorticoid Signaling System

Glucocorticoids regulate many crucial biologic functions through their cytoplasmic/nuclear glucocorticoid receptors (GR). Excess, deficiency, or alteration in tissue sensitivity to glucocorticoids has been associated with major causes of human morbidity and mortality. Brx, a cytoplasmic Rho family guanine nucleotide exchange factor, binds to and influences the activity of several nuclear hormone receptors. We examined the functional and molecular interactions between GR and Brx. The glucocorticoid sensitivity of lymphocytes obtained from mice haplo-insufficient for Brx was significantly decreased. Conversely, GR-mediated transcriptional activity of a glucocorticoid response element (GRE)-mediated glucocorticoid-responsive promoter was enhanced by Brx in a guanine nucleotide exchange factor domain-dependent fashion. Brx interacted with GR, forming a ternary complex with RhoA. In a chromatin immunoprecipitation assay, Brx and RhoA were co-precipitated with GREs only in the presence of ligand-activated GR. Extracellularly administered lysophosphatidic acid, which activates its signaling cascade through a specific membrane GTP-binding protein (G-protein)-coupled receptor in a G-protein alpha(13)-, Brx-, and RhoA-dependent fashion, enhanced GR transcriptional activity, whereas depletion of endogenous Brx attenuated this effect. These findings suggest that glucocorticoid signaling and, hence, the tissue sensitivity to glucocorticoids, may be coupled to extracellular signals via Brx and small G-proteins. Nuclear Brx might act as a local GRE-GR-transcriptosome activator by mediating the effect of small G-proteins on glucocorticoid-regulated genes.

A Novel Mechanism of G Protein-dependent Phosphorylation of Vasodilator-stimulated Phosphoprotein

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate of protein kinase A (PKA). Here we described the novel mechanism of VASP phosphorylation via cAMP-independent PKA activation. We showed that in human umbilical vein endothelial cells (HUVECs) alpha-thrombin induced phosphorylation of VASP. Specific inhibition of Galpha13 protein by the RGS domain of a guanine nucleotide exchange factor, p115RhoGEF, inhibited thrombin-dependent phosphorylation of VASP. More importantly, Galpha13-induced VASP phosphorylation was dependent on activation of RhoA and mitogen-activated protein kinase kinase kinase, MEKK1, leading to the stimulation of the NF-kappaB signaling pathway. alpha-Thrombin-dependent VASP phosphorylation was inhibited by small interfering RNA-mediated knockdown of RhoA, whereas Galpha13-dependent VASP phosphorylation was inhibited by a specific RhoA inhibitor botulinum toxin C3 and by a dominant negative mutant of MEKK1. We determined that Galpha13-dependent VASP phosphorylation was also inhibited by specific PKA inhibitors, PKI and H-89. In addition, the expression of phosphorylation-deficient IkappaB and pretreatment with the proteasome inhibitor MG-132 abolished Galpha13- and alpha-thrombin-induced VASP phosphorylation. In summary, we have described a novel pathway of Galpha13-induced VASP phosphorylation that involves activation of RhoA and MEKK1, phosphorylation and degradation of IkappaB, release of PKA catalytic subunit from the complex with IkappaB and NF-kappaB, and subsequent phosphorylation of VASP.

Pituitary Adenylyl Cyclase-activating Polypeptide 38 Reduces Astroglial Proliferation by Inhibiting the GTPase RhoA

Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) plays an important role in the proliferation and differentiation of neural cells. In the present study, we have investigated how PACAP38 inhibits the proliferation of cultured neocortical astroglial cells. When applied to synchronized cells during the G(1) phase of the cell cycle, PACAP38 diminished the subsequent nuclear uptake of bromodeoxyuridine. When applied for 2 days, it reduced the cell number. PACAP38 did not exert its antiproliferative effect by activating protein kinase A. It also did not reduce the activity of mitogen-activated protein kinases essential for G(1) phase progression. Instead, PACAP38 acted on a member of the Rho family of small GTPases. It reduced the activity of RhoA as was shown with a Rhotekin pull-down assay. The decrease in endogenous RhoA activity induced by treatment of the cells with C3 exotoxin or by expression of dominant negative RhoA also reduced the nuclear uptake of bromodeoxyuridine. In contrast, expression of constitutively active RhoA prevented the effect of PACAP38. Our data show a novel signal transduction pathway by which the neuropeptide influences cell proliferation.

Outgrowth of Neurites from NIE-115 Neuroblastoma Cells Is Prevented on Repulsive Substrates through the Action of PAK

In the central nervous system (CNS), damaged axons are inhibited from regeneration by glial scars, where secreted chondroitin sulfate proteoglycan (CSPG) and tenascin repulse outgrowth of neurites, the forerunners of axons and dendrites. During differentiation, these molecules are thought to form boundaries for guiding neurons to their correct targets. In neuroblastoma NIE-115 cells, outgrowth of neurites on laminin could be induced by serum starvation or inhibition of RhoA by Clostridium botulinum C3 toxin. The outgrowing neurites avoided crossing onto the repulsive substrate CSPG or tenascin. This avoidance response was partially overcome on expression of membrane-targeted and kinase-inactive forms of PAK. In these cells, the endogenous PAK isoforms colocalized with actin in distinctive sites, alphaPAK in the cell center as small clusters and along the neurite shaft and betaPAK and gammaPAK in areas with membrane ruffles and filopodia, respectively. When isoform-specific N-terminal PAK sequences were introduced to interfere with PAK function, substantially more neurites crossed onto CSPG when cells contained a gammaPAK-derived peptide but not the corresponding alphaPAK- or betaPAK-derived peptide. Thus, while neurite outgrowth can be promoted by RhoA inhibition, overcoming the accompanying repulsive guidance response will require modulation of PAK activity. These results have therapeutic implications for CNS repair processes.

Analysis of Gene Expression in Rat Lacrimal Gland Duct Cells using Laser Capture Microdissection and cDNA Microarrays

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABAA receptor channels. In a recent study, we showed that Zn2+ regulates GABAA reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn2+ initiates this process by activating the Gq-coupled metabotropic Zn2+ receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca2+ indicator and exposing cells to Zn2+, which triggered consistent Ca2+ responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn2+ treatment under these conditions did not increase the intracellular concentrations of Zn2+ itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH4+ influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn2+ pretreatment induced a Ca2+-dependent increase in KCC2 activity. The effects of Zn2+ on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39−/− mice, suggesting that Zn2+ acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn2+-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn2+. These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn2+ stimulation of mZnR/GPR39.

Inhibition of ROMK potassium channel by syntaxin 1A.

ROMK potassium channels are present in the cortical collecting duct (CCD) of the kidney and serve as apical exit pathways for K+ secretion in this nephron segment. K+ secretion in the CCD is regulated by multiple factors. In this study, we show that syntaxin 1A, but not syntaxin 3 or 4, inhibited whole cell ROMK currents in Xenopus laevis oocytes. Syntaxin 1A, but not syntaxin 3 or 4, interacted with the COOH-terminal cytoplasmic domain of ROMK in intro. Coexpression with synaptobrevin 2 reversed inhibition of whole cell ROMK currents by syntaxin 1A. In excised inside-out membranes of oocytes, application of fusion proteins containing the cytoplasmic region of syntaxin 1A to the cytoplasmic face caused a dose-dependent inhibition of ROMK. We further examined regulation of the K+ channels in the CCD by syntaxin 1A. Application of botulinum toxin C1 to the excised inside-out membranes of the CCD caused an increase in the activity of K+ channels. In contrast, application of toxin B had no effects. These results suggest that syntaxin 1A causes a tonic inhibition of the K+ channels in the apical membrane of the CCD. Binding of synaptobrevin 2 to syntaxin 1A during docking and fusion of transport vesicles to the plasma membranes of CCD may lead to activation of these channels.

External pH changes affect NMDA-evoked and spontaneous release of cholecystokinin, somatostatin and noradrenaline from rat cerebrocortical nerve endings

It was previously reported that the K +-evoked release of somatostatin-like immunoreactivity (SRIF-LI) and of cholecystokinin-like immunoreactivity (CCK-LI) from superfused rat cerebrocortical synaptosomes can be enhanced by NMDA or d-serine alone. We here studied the effects of extraterminal pH changes on SRIF-LI and CCK-LI release. Lowering pH from 7.4 to 6.9 or 6.4 abolished the effects of NMDA or d-serine on the K +-evoked peptide release. Identical results were obtained when external pH was raised to 8 or 8.7. Sudden alkalinization of the superfusion medium, in absence of K +-depolarization, induced SRIF-LI or CCK-LI release which was insensitive to NMDA. Based on experiments in Ca 2+-free medium and with voltage-sensitive Ca 2+ channel (VSCC) blockers, the pH 8.7-induced release of SRIF-LI and CCK-LI was only in part (30–50%) dependent on external Ca 2+ and Ca 2+ channel activation. In contrast, the alkalinization-evoked release of [ 3 H ]noradrenaline was highly sensitive to external Ca 2+ removal and to blockade of Ca 2+ channels with ω-conotoxins. The pH 8.7-evoked SRIF-LI and CCK-LI was about halved in synaptosomes intoxicated with botulinum toxin C1. The results suggest that the pH-sensitive NMDA receptors mediating somatostatin and cholecystokinin release contain NR1 subunits lacking the exon-5 cassette. Alkalinization represents a novel releasing stimulus which elicits neuropeptide release in part by conventional exocytosis and largely by an external Ca 2+-independent mechanism. Differently, the release of noradrenaline provoked by alkalinization occurs entirely by conventional exocytosis.

External pH changes affect NMDA-evoked and spontaneous release of cholecystokinin, somatostatin and noradrenaline from rat cerebrocortical nerve endings

It was previously reported that the K + -evoked release of somatostatin-like immunoreactivity (SRIF-LI) and of cholecystokinin-like immunoreactivity (CCK-LI) from superfused rat cerebrocortical synaptosomes can be enhanced by NMDA or d -serine alone. We here studied the effects of extraterminal pH changes on SRIF-LI and CCK-LI release. Lowering pH from 7.4 to 6.9 or 6.4 abolished the effects of NMDA or d -serine on the K + -evoked peptide release. Identical results were obtained when external pH was raised to 8 or 8.7. Sudden alkalinization of the superfusion medium, in absence of K + -depolarization, induced SRIF-LI or CCK-LI release which was insensitive to NMDA. Based on experiments in Ca 2+ -free medium and with voltage-sensitive Ca 2+ channel (VSCC) blockers, the pH 8.7-induced release of SRIF-LI and CCK-LI was only in part (30–50%) dependent on external Ca 2+ and Ca 2+ channel activation. In contrast, the alkalinization-evoked release of [ 3 H ]noradrenaline was highly sensitive to external Ca 2+ removal and to blockade of Ca 2+ channels with ω-conotoxins. The pH 8.7-evoked SRIF-LI and CCK-LI was about halved in synaptosomes intoxicated with botulinum toxin C1. The results suggest that the pH-sensitive NMDA receptors mediating somatostatin and cholecystokinin release contain NR1 subunits lacking the exon-5 cassette. Alkalinization represents a novel releasing stimulus which elicits neuropeptide release in part by conventional exocytosis and largely by an external Ca 2+ -independent mechanism. Differently, the release of noradrenaline provoked by alkalinization occurs entirely by conventional exocytosis.

Clostridium botulinum C2 toxin – New insights into the cellular up-take of the actin-ADP-ribosylating toxin

AbstractClostridium botulinum C2 toxin is a member of the family of binary actin-ADP-ribosylating toxins. It consists of the enzyme component C2I, and the separated binding/translocation component C2II. Proteolytically activated C2II forms heptamers and binds to a carbohydrate cell surface receptor. After attachment of C2I, the toxin complex is endocytosed to reach early endosomes. At low pH of endosomes, C2II-heptamers insert into the membrane, form pores and deliver C2I into the cytosol. Here, C2I ADP-ribosylates actin at Arg177 to block actin polymerization and to induce depolymerization of actin filaments. The mini-review describes main properties of C2 toxin and discusses new findings on the involvement of chaperones in the up-take process of the toxin.

The actin-ADP-ribosylating Clostridium botulinum C2 toxin

Clostridium botulinum C2 toxin is the prototype of actin-ADP-ribosylating toxins. The toxin consists of the enzyme component C2I and the separated binding/translocation component C2II. C2II is proteolytically activated to form heptamers, which bind the enzyme component. After endocytosis of the receptor–toxin complex, the enzyme component enters the cytosol from an acidic endosomal compartment to modify G-actin at arginine177. Recent data indicate that chaperons are involved in the translocation process of the toxin.

Cellular Uptake of Clostridium botulinum C2 Toxin: Membrane Translocation of a Fusion Toxin Requires Unfolding of Its Dihydrofolate Reductase Domain

The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.

Clostridium botulinum C2 Toxin: LOW pH-INDUCED PORE FORMATION IS REQUIRED FOR TRANSLOCATION OF THE ENZYME COMPONENT C2I INTO THE CYTOSOL OF HOST CELLS

The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II (80 kDa) and the enzyme component C2I, which ADP-ribosylates G-actin in the cytosol of cells. Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells. Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa. We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells. Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse. Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium (pH < 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium. Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes. When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore. A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation. Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially. C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH. When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased. Overall, C2IIa pores were essential but not sufficient for translocation of C2I. Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.

The Host Cell Chaperone Hsp90 Is Essential for Translocation of the Binary Clostridium botulinum C2 Toxin into the Cytosol

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool.

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.

Syntaxin I modulation of presynaptic calcium channel inactivation revealed by botulinum toxin C1.

The chick ciliary ganglion calyx-type nerve terminal was used to examine voltage-sensitive inactivation of presynaptic N-type Ca2+ channels and to test if this inactivation is modulated by the transmitter release-associated protein syntaxin I. We tested the role of this protein with botulinum toxin C1 (BtC1) which cleaves syntaxin I close to its membrane anchor. The presynaptic Ca2+ current inactivated as two distinct populations with approximately 75% inactivating at a depolarized potential, V1/2 approximately -15 mV, with the remainder inactivating at approximately -75 mV. BtC1 had no detectable effect on the latter component but resulted in a approximately 7 mV positive shift in the V1/2 of the -15 mV inactivating component. These results confirm that the bulk of presynaptic N-type Ca2+ channels are in general resistant to voltage dependent inactivation and provide the first direct evidence that the physiological properties of presynaptic nerve terminal Ca2+ channels are subject to modulation by release site-associated proteins.

CAMP-induced AQP2 translocation is associated with RhoA inhibition through RhoA phosphorylation and interaction with RhoGDI.

We have recently demonstrated that inhibition of Rho GTPase with Clostridium difficile toxin B, or with Clostridium botulinum C3 toxin, causes actin depolymerization and translocation of aquaporin 2 (AQP2) in renal CD8 cells in the absence of hormonal stimulation. Here we demonstrate that Rho inhibition is part of the signal transduction cascade activated by vasopressin leading to AQP2 insertion into the apical membrane. Quantitation of active RhoA (GTP-bound) by selective pull down experiments demonstrated that the amount of active RhoA decreased upon stimulation of CD8 cells with the cAMP-elevating agent forskolin. Consistent with this observation, forskolin treatment resulted in a decreased expression of membrane-associated (active) Rho, as assessed by cell fractionation followed by western blotting analysis. In addition, the abundance of the endogenous Rho GDP dissociation inhibitor (Rho-GDI) was found to have decreased in the membrane fraction after forskolin stimulation. Co-immunoprecipitation experiments revealed that, after forskolin stimulation, the amount of Rho-GDI complexed with RhoA increased, suggesting that Rho GTPase inhibition occurs through association of RhoA with Rho-GDI. Finally, forskolin stimulation was associated with an increase in Rho phosphorylation on a serine residue, a protein modification known to stabilize the inactive form of RhoA and to increase its interaction with Rho-GDI. Taken together, these data demonstrate that RhoA inhibition through Rho phosphorylation and interaction with Rho-GDI is a key event for cytoskeletal dynamics controlling cAMP-induced AQP2 translocation.

Effects of H2O2on Microfilaments of Cultured Endothelial Cells

Hydrogen peroxide (H2O2) is an important granulocyte derived mediator of endothelial cell injury. Alterations of the microfilaments system (especially of actin) in endothelial cells may be relevant for the pathogenesis of vascular leakage. In the study presented effects of H2O2 on actin monomers (G-actin) and filamentous actin (F-actin) were examined in cultured pulmonary artery endothelial cells. Phalloidin which blocks actin depolymerization by inhibiting actin monomer dissociation and C. Botulinum C2 toxin which ADP-ribosylates G-actin thereby inhibiting actin polymerization were used as tools for the study of H2O2–related actin alterations. Exposure of cells to 2 mM H2O2 resulted in a biphasic change of F-actin with an early decrease (15 min) and a subsequent doubling (120 min) paralleled by an inverse G-actin pattern. In endothelial cells with a 20% reduction of F-actin-brought about by preincubation with C2-toxin for 150 min- H2O2-related actin polymerization was unimpaired. In cells with completely dissolved F-actin (Bot. C2-toxin for 210 min) no actin polymerization occurred upon H2O2 application. Phalloidin blocked the early H2O2-induced F-actin decrease and slowed down late actin polymerization. Effects of H2O2 on endothelial actin were abolished in the presence of scavengers of oxygen metabolites (catalase) and by the poly (ADP) ribose-polymerase inhibitor aminobenzamid. The data presented are compatible with the concept that H2O2 stimulates actin turnover and actin nucleation which—in the long run—result in H2O2 related formation of new actin filaments. This process was blocked by oxygen metabolite scavengers and by inhibition of DNA strand break repair mechanisms.

Protein tyrosine phosphatase PTP20 induces actin cytoskeleton reorganization by dephosphorylating p190 RhoGAP in rat ovarian granulosa cells stimulated with follicle-stimulating hormone.

We identified 25 protein tyrosine phosphatases (PTPs) expressed in rat ovarian granulosa cells. Of these PTPs, the expression levels of at least PTP20, PTP-MEG1, PTPepsilonM, and PTPepsilonC significantly changed during the estrous cycle. We examined the cellular functions of PTP20 in granulosa cells by expressing the wild type, a catalytically inactive CS mutant in which Cys229 of PTP20 was changed to Ser, or a substrate-trapping DA mutant in which Asp197 was mutated to Ala, using an adenovirus vector. Overexpression of the wild type, but not of the CS mutant, induced retraction of the cell body with the extension of long, dendritic-like processes after stimulation with FSH, a critical factor for the survival and differentiation of these cells. In addition, cell adhesion to the substratum decreased in an FSH-dependent manner. Inhibiting Rho GTPase activity with C3 botulinum toxin caused similar morphological changes. The FSH-enhanced phosphotyrosine (p-Tyr) level of p190 RhoGAP was selectively reduced by the overexpressed wild type, but not by mutated PTP20. Although p190 RhoGAP is tyrosine phosphorylated by c-Src via the tyrosine kinase Pyk2, wild-type PTP20 had little effect on p-Tyr418 of c-Src and no effect on p-Tyr402 of Pyk2, which are required for full c-Src activity and for interacting between Pyk2 and c-Src, respectively. The CS and DA mutants as well as the wild type reduced the formation of p190 RhoGAP-p120 RasGAP complexes. Confocal microscopy analysis revealed that PTP20 intracellularly colocalizes with p190 RhoGAP. These results demonstrate that PTP20 regulates the functions of granulosa cells in an FSH-dependent manner by dephosphorylating p190 RhoGAP and subsequently inducing reorganization of the actin cytoskeleton. Moreover, our data suggest that PTPs play significant roles in controlling the dynamics of ovarian functions.

Galphaq signaling is required for Rho-dependent transcriptional activation of the cyclooxygenase-2 promoter in fibroblasts.

Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase-2 (COX-2) expression through a Rho-dependent, protein kinase C (PKC)-independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562-27566). However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) that are coupled to the GRP receptor in Rho-dependent COX-2 expression has not been elucidated. In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock-outs (DKO) for Galpha(q/11) and Galpha(12/13) to demonstrate that COX-2 promoter activation by GRP requires Galpha(q). Furthermore, we show that GRP-dependent COX-2 gene expression, as assessed by a COX-2 reporter luciferase assay, was induced in cells lacking Galpha(12/13) but was blocked in cells that did not express Galpha(q/11). GRP-dependent COX-2 promoter induction in Galpha(q/11) deficient cells was rescued by expression of wild type Galpha(q) but blocked by inhibition of calcium signaling in calcium-free media or in cells treated with 2-aminoethoxydiphenylborate (2-APB). Co-stimulation of transfected Galpha(q/11) deficient cells with GRP and thapsigargin (TG) induced the COX-2 promoter. Activation of endogenous Rho by expression of Onco-lbc or expression of Rho A Q63L resulted in COX-2 promoter activation in Galpha(q/11) deficient cells. Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX-2 promoter induction. Expression of Galpha(q) Q209L in the well-characterized fibroblast cell line, NIH3T3, induced the COX-2 promoter which was blocked by expression of C3 toxin. These results demonstrate that calcium signaling mediated by Galpha(q) and Rho play critical roles in GRP-dependent COX-2 expression in fibroblasts.