The Lbc Rho Guanine Nucleotide Exchange Factor/α-Catulin Axis Functions in Serotonin-induced Vascular Smooth Muscle Cell Mitogenesis and RhoA/ROCK Activation

Serotonin (5-hydroxytryptamine, 5-HT) is mitogenic for several cell types including pulmonary arterial smooth muscle cells (PASMC), and is associated with the abnormal vascular smooth muscle remodeling that occurs in pulmonary arterial hypertension. RhoA/Rho kinase (ROCK) function is required for 5-HT-induced PASMC mitogenesis, and 5-HT activates RhoA; however, the signaling steps are poorly defined. Rho guanine nucleotide exchange factors (Rho GEFs) transduce extracellular signals to Rho, and we found that 5-HT treatment of PASMC led to increased membrane-associated Lbc Rho GEF, suggesting modulation by 5-HT. Lbc knockdown by siRNA attenuated 5-HT-induced thymidine uptake in PASMC, indicating a role in PASMC mitogenesis. 5-HT triggered Rho-dependent serum response factor-mediated reporter activation in PASMC, and this was reduced by Lbc depletion. Lbc knockdown reduced 5-HT-induced RhoA/ROCK activation, but not p42/44 ERK MAP kinase activation, suggesting that Lbc is an intermediary between 5-HT and RhoA/ROCK, but not ERK. 5-HT stimulation of PASMC led to increased association between Lbc, RhoA, and the α-catulin scaffold. Furthermore, α-catulin knockdown attenuated 5-HT-induced PASMC thymidine uptake. 5-HT-induced PASMC mitogenesis was reduced by dominant-negative G_q protein, suggesting cooperation with Lbc/α-catulin. These results for the first time define a Rho GEF involved in vascular smooth muscle cell growth and serotonin signaling, and suggest that Lbc Rho GEF family members play distinct roles. Thus, the Lbc/α-catulin axis participates in 5-HT-induced PASMC mitogenesis and RhoA/ROCK signaling, and may be an interventional target in diseases involving vascular smooth muscle remodeling.     

GDP-bound and Nucleotide-free Intermediates of the Guanine Nucleotide Exchange in the Rab5·Vps9 System

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg²⁺ and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.     

The Guanine Nucleotide Exchange Factor RIC8 Regulates Conidial Germination through Gα Proteins in Neurospora crassa

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.     

Protective Effect of Bajijiasu Against β-Amyloid-Induced Neurotoxicity in PC12 Cells

Beta-amyloid peptide (Aβ), a major protein component of senile plaques associated with Alzheimer’s disease (AD), is also directly neurotoxic. Mitigation of Aβ-induced neurotoxicity is thus a possible therapeutic approach to delay or prevent onset and progression of AD. This study evaluated the protective effect of Bajijiasu (β- d-fructofuranosyl (2–2) β- d-fructofuranosyl), a dimeric fructose isolated from the Chinese herb Radix Morinda officinalis, on Aβ-induced neurotoxicity in pheochromocytoma (PC12) cells. Bajijiasu alone had no endogenous neurotoxicity up to 200 μM. Brief pretreatment with 10–40 μM Bajijiasu (2 h) significantly reversed the reduction in cell viability induced by subsequent 24 h exposure to Aβ_25–35 (21 μM) as measured by MTT and LDH assays, and reduced Aβ_25–35-induced apoptosis as indicated by reduced annexin V-EGFP staining. Bajijiasu also decreased the accumulation of intracellular reactive oxygen species and the lipid peroxidation product malondialdehyde in PC12 cells, upregulated expression of glutathione reductase and superoxide dismutase, prevented depolarization of the mitochondrial membrane potential (Ψm), and blocked Aβ_25–35-induced increases in [Ca²⁺] _i . Furthermore, Bajijiasu reversed Aβ_25–35-induced changes in the expression levels of p21, CDK4, E2F1, Bax, NF-κB p65, and caspase-3. Bajijiasu is neuroprotective against Aβ_25–35-induced neurotoxicity in PC12 cells, likely by protecting against oxidative stress and ensuing apoptosis.     

The Role of Heat Shock Protein 70 in the Protective Effect of YC-1 on β-Amyloid-Induced Toxicity in Differentiated PC12 Cells

Neurodegenerative brain disorders such as Alzheimer’s disease (AD) have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70) plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC), on Aβ_25–35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aβ_25–35 (10 µM) significantly increased p25 protein production in a pattern that was consistent with the increase in μ-calpain expression. Moreover, Aβ_25–35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5–10 µM) prevented the cell death induced by Aβ_25–35. In addition, YC-1 (1, 10 µM) significantly blocked Aβ_25–35-induced μ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5–20 µM) alone or combined with Aβ_25–35 (10 µM) significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM) or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM) did not affect the neuroprotective effect of YC-1 against Aβ_25–35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aβ_25–35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.     

Protective Effect of 1-Methylated β-Carbolines Against 3-Morpholinosydnonimine-Induced Mitochondrial Damage and Cell Viability Loss in PC12 Cells

The present study investigated the effect of 1-methylated beta-carbolines (harmaline, harmalol and harmine) on change in the mitochondrial membrane permeability and cell death due to reactive nitrogen species in differentiated PC12 cells. beta-Carbolines, caspase inhibitors (z-LEHD.fmk and z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell viability loss due to 3-morpholinosydnonimine (SIN-1) in PC12 cells. beta-Carbolines inhibited the nuclear damage, the decrease in mitochondrial transmembrane potential, the cytochrome c release, the formation of reactive oxygen species and the depletion of GSH caused by SIN-1 in PC12 cells. beta-Carbolines decreased the SIN-1-induced formations of 3-nitrotyrosine, malondialdehyde and carbonyls in PC12 cells. The results show that 1-methylated beta-carbolines attenuate SIN-1-induced mitochondrial damage. This results in the inhibition of caspase-9 and -3 and apoptotic cell death in PC12 cells by suppressing the toxic actions of reactive oxygen and nitrogen species, including the GSH depletion.     

Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described. More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation of the Rac 1/PYGM pathway. IL-2-stimulated serine phosphorylation was corroborated in Kit 225 T cells cultures. A parallel pharmacological and genetic approach identified PKCθ as the serine/threonine kinase responsible for αPIX serine phosphorylation. The phosphorylated state of αPIX was required to activate first Rac 1 and subsequently PYGM. These results demonstrate that the IL-2 receptor activation, among other early events, leads to activation of PKCθ. To activate Rac 1 and consequently PYGM, PKCθ phosphorylates αPIX in T cells. The biological significance of this PKCθ/αPIX/Rac 1 GTPase/PYGM signaling pathway seems to be the control of different cellular responses such as migration and proliferation.     

Protective Effects of Hydroxysafflor Yellow A on β-Amyloid-Induced Neurotoxicity in PC12 Cells

The accumulation of extracellular amyloid-β peptide (Aβ) has been considered as one of the important causes of Alzheimer’s disease (AD), the most prevalent form of dementia. Hydroxysafflor yellow A (HSYA), a major active chemical component isolated from Carthamus tinctorius L., has been shown to possess neuroprotective actions in various ischemic models in vivo. The present study aimed to investigate the potential protective effect of HSYA against Aβ-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The PC12 cells were pretreated with different concentrations (20, 40 and 80 μM) of HSYA for 2 h and then further treated with Aβ (20 μM) for 24 h. The results showed that Aβ could significantly decrease cell viability, glutathione level, mitochondrial membrane potential and the ratio of Bcl-2/Bax protein expression, while elevate the release of lactate dehydrogenase, the formation of DNA fragmentation, the levels of malondialdehyde and intracellular reactive oxygen species in PC12 cells. However, pretreatment with HSYA could effectively reverse these changes induced by Aβ in PC12 cells. Our experimental results demonstrate that HSYA may be a potential neuroprotective agent warranting further development for treatment of AD.     

βArrestin1 Regulates the Guanine Nucleotide Exchange Factor RasGRF2 Expression and the Small GTPase Rac-mediated Formation of Membrane Protrusion and Cell Motility

βArrestin proteins shuttle between the cytosol and nucleus and have been shown to regulate G protein-coupled receptor signaling, actin remodeling, and gene expression. Here, we tested the hypothesis that βarrestin1 regulates actin remodeling and cell migration through the small GTPase Rac. Depletion of βarrestin1 promotes Rac activation, leading to the formation of multipolar protrusions and increased cell circularity, and overexpression of a dominant negative form of Rac reverses these morphological changes. Small interfering RNA library screen identifies RasGRF2 as a target of βarrestin1. RasGRF2 gene and protein expression levels are elevated following depletion of βarrestin1, and the consequent activation of Rac results in dephosphorylation of cofilin that can promote actin polymerization and formation of multipolar protrusions, thereby retarding cell migration and invasion. Together, these results suggest that βarrestin1 regulates rasgrf2 gene expression and Rac activation to affect membrane protrusion and cell migration and invasion.     

Temporal and Hormonal Control of β-1,3-Glucanase in Phaseolus vulgaris L

The endo-beta-1, 3-glucanase (beta-1, 3-glucan 3-glucanhydrolase, EC 3.2.1.6) extracted from Phaseolus vulgaris L. cv. Red Kidney had a pH optimum of 5 and a temperature optimum of 50 C. Excision of plant tissue resulted in an increase in beta-1, 3-glucanase activity after a 6-hour lag period. The increase could be prevented by indole-3-acetic acid, gibberellic acid, and cytokinins. Ethylene (half-maximal concentration = 0.1 microliter/liter) promoted the synthesis of beta-1, 3-glucanase, and 10% CO(2) overcame some of the ethylene effect. Cycloheximide prevented the induction of beta-1, 3-glucanase, but actinomycin D and chromomycin A(3) had only a partial effect.The amount of callose in sieve tube cells correlated with levels of beta-1, 3-glucanase, suggesting that this enzyme played a role in the degradation of beta-1, 3-glucans.     

Inhibition of SIN-1–Induced Change in Mitochondrial Membrane Permeability in PC12 Cells by Dopamine

Dopamine (50 or 100 microM) attenuated the nuclear damage and cell death due to 500 microM SIN-1, a donor of superoxide and nitric oxide, in differentiated PC12 cells whereas 200 microM dopamine did not depress cell death. Dopamine at 50-100 microM for a 4-h treatment did not show a significant cytotoxic effect on PC12 cells. Dopamine (100 microM) inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, activation of caspase-3, formation of reactive oxygen species, and depletion of glutathione (GSH) due to 500 microM SIN-1 in PC12 cells. The reaction of dopamine with peroxynitrite reduced an amount of peroxynitrite. The results suggest that dopamine exhibits a biphasic effect against the cytotoxicity of SIN-1 depending on concentrations. Dopamine at 50-100 microM may attenuate the reactive nitrogen species-induced viability loss in PC12 cells by suppressing the mitochondrial membrane permeability change through inhibition of the formation of reactive species, including peroxynitrite.     

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Abstract

Rat C6 glioma cells have both β₁- and β₂-adrenergic receptors in ～ 7:3 ratio. When the cells were exposed to the β-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [³H] CGP-12177 to intact cells. Using the β₂-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both β₁ and β₂ receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of ～ 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [¹²⁵I] iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous β₁-and β₂-adrenergic receptors in the same cell line.     

The ultrastructural effects of β-amyloid peptide on cultured PC12 cells: changes in cytoplasmic and intramembranous features.

Summary The fine structural features of cultured PC12 cells were investigated after treatment for 1, 3, or 5 days with different concentrations of the vascular form of β- 1–40 (β-AP). PC12 cells treated with β-AP showed time- and concentration-dependent lysosomal system activation and cell toxicity. We observed increases in the number and size of cytoplasmic lysosomes as indicated by increased acid phosphatase reactivity. Some lysosomes were in the form of multivesicular bodies or large residual bodies that appeared to arise by autophagia or by endocytotic uptake. Double-sided plasma membrane invaginations were observed to give rise to increasingly extensive intracytoplasmic vacuolization that was correlated with duration of β-AP treatment. Freeze-fracture studies of the intramembranous particle (IMP) population in the plasma membrane P-face showed that both control and β-AP treated cells had two major P-face IMP populations, small-diameter (4–8 nm) IMPs, and large-diameter (≤ 9nm) IMPs. The larger category of IMPs was found to possess a greater average diameter in the β-AP treated cells than in the control cells. These IMPs could represent modifications to existing transmembranous receptors, channels, or transducing molecules by the β-AP. These results demonstrate that β-AP can induce time- and concentration-dependent ultrastructural changes in PC12 cell membranes.     

Synergistic protective effect of paeoniflorin and β-ecdysterone against rotenone-induced neurotoxicity in PC12 cells

There are several factors, like oxidative stress and neurons loss, involving neurodegenerative diseases such as Parkinson’s disease (PD). The combination of antioxidant and anti-apoptotic agent is becoming a promising approach to fight against PD. This study evaluates the hypothesis that paeoniflorin (PF) and β-ecdysterone (β-Ecd) synergize to protect PC12 cells against toxicity induced by PD-related neurotoxin rotenone. The combination of PF and β-Ecd, hereafter referred to as the PF/β-Ecd, at suboptimal concentrations increased the viability of rotenone-exposed PC12 cells in a synergistic manner. PF and β-Ecd cooperate to attenuate the rotenone-induced apoptosis by decrease in Bax expression, caspase-9 activity, and caspase-3 activity. PF or PF/β-Ecd, but not β-Ecd, inhibited rotenone-triggered protein kinase C-δkinase C-δ (PKCδ) upregulation and nuclear factor κB (NF-κB) activation. β-Ecd or PF/β-Ecd, but not PF, enhanced serine/threonine protein kinase (Akt) activation, promoted nuclear factor E2-related factor 2 (Nrf2) nuclear accumulation, suppressed reactive oxygen species (ROS) production. Neuroprotection of PF/β-Ecd could be completely blocked by PKCδ inhibitor rottlerin plus Akt specific inhibitor LY294002. Dual blockade of the PKCδ/NF-κB pathway by PF and activation of Akt/Nrf2 pathway by β-Ecd results in a synergistic neuroprotective effect against rotenone-induced neurotoxicity in vitro. These findings provide the rationale for determining the in vivo activity of combined therapy with PF and β-Ecd against PD.     

Laminin-1 and the RKRLQVQLSIRT Laminin-1 α1 Globular Domain Peptide Stimulate Matrix Metalloproteinase Secretion by PC12 Cells

Here we have investigated the ability of laminin-1 and specific laminin-1-derived synthetic peptides to stimulate neuronal cell matrix metalloproteinase secretion. Zymographic analysis of conditioned media from laminin-1-treated PC12 and NG108-15 cells revealed a 72-kDa matrix metalloproteinase which was not secreted by untreated cells. Laminin-1 α1 chain-derived synthetic peptides, AASIKVAVSADR (LAM-L) and RKRLQVQLSIRT (AG-73), also stimulated PC12 cell secretion of a 72-kDa matrix metalloproteinase. We further investigated the structural requirements of AG-73 for cell attachment, neurite outgrowth, and matrix metalloproteinase secretion using a series of AG-73 analogs that had single amino acids substituted with alanine. At the substrate levels tested, the AG-73 peptide promoted the adhesion of 67% of the PC12 cells and neurite outgrowth in 71% of the PC12 cells. Substitutions in any one of the amino acids within the central LQVQ sequence resulted in a large reduction in cell attachment whereas substitution in the carboxyl terminal proximal amino acids L, S, and R had little effect on attachment. Alanine substitution of any of the amino terminal proximal LQV amino acids and the carboxyl terminal L, I, and R residues resulted in a 65–91% reduction in neurite outgrowth. These data demonstrate that the sequence requirements for cell attachment and neurite outgrowth were not necessarily coupled but that the sequence requirements for neurite outgrowth and matrix metalloproteinase secretion were identical. We conclude that laminin-1 is able to stimulate neuronal cells to secrete a matrix metalloproteinase. Further, this study identifies the LQVXLXIR laminin-1 α1 globular domain peptide to be capable of stimulating both neurite outgrowth and matrix metalloproteinase secretion.     

Hydrogen sulfide reduces mRNA and protein levels of β-site amyloid precursor protein cleaving enzyme 1 in PC12 cells

Hydrogen sulfide (H(2)S) is now identified as a new neuromodulator. Increasing evidence suggest that H(2)S may play an important role in the progression of Alzheimer's disease (AD). The aim of the present study is to investigate the effects of H(2)S on beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) expression and amyloid beta (Aβ) secretion in PC12 cells. The levels of BACE-1 mRNA were measured by quantitative polymerase chain reaction analysis. BACE-1 protein levels were assessed by Western blot. Cellular culture medium levels of Aβ1-42 were analyzed by ELISA. We found that sodium hydrosulfide (NaHS), a H(2)S donor, decreased BACE-1 mRNA and protein levels and Aβ1-42 release. Furthermore, NaHS promoted the phosphorylation of Akt and ERK but not JNK or p38 MAPK. However, the effects of NaHS on BACE-1 expression and Aβ1-42 secretion were abolished by inhibitors of phosphatidylinositol 3-kinase (PI3-K), but not of mitogen-activated protein kinase kinases (MEK). Our data indicate that H(2)S reduces BACE-1 expression in PC12 cells via activation of PI3-K/Akt signaling pathways. H(2)S releasing drugs may have therapeutic potential in AD patients.