Mediated Signal Transduction Pathways Research Articles

Drug Development Targeting the Glycogen Synthase Kinase-3β (GSK-3β)-Mediated Signal Transduction Pathway: The Role of GSK-3β in the Maintenance of Steady-State Levels of Insulin Receptor Signaling Molecules and Nav1.7 Sodium Channel in Adrenal Chromaffin Cells

Glycogen synthase kinase-3 (GSK-3) is constitutively active in nonstimulated cells, where the majority of its substrates undergo inactivation/proteolysis by phosphorylation. Extracellular stimuli (e.g., insulin) catalyze inhibitory Ser9-phosphorylation of GSK-3β, turning on signaling and causing other biological consequences otherwise constitutively suppressed by GSK-3β. Regulated and dysregulated activities of GSK-3β are pivotal to health, disease, and therapeutics (e.g., insulin resistance, neurodegeneration, tumorigenesis, inflammation); however, the underlying mechanisms of multifunctional GSK-3β remain elusive. In cultured bovine adrenal chromaffin cells, 1) constitutive and negatively-regulated activities of GSK-3β up- and down-regulated insulin receptor, insulin receptor substrate-1 (IRS-1), IRS-2, and Akt levels via controlling proteasomal degradation and protein synthesis; 2) nicotinic receptor/protein kinase C-α (PKC-α) / extracellular signal-regulated kinase (ERK) pathway up-regulated IRS-1 and IRS-2 levels, enhancing insulin-induced the phosphoiNOSitide 3-kinase (PI3K) / Akt / GSK-3β pathway; 3) inhibition of calcineurin by cyclosporin A or FK506 down-regulated IRS-2 level, attenuating insulin-like growth factor-I (IGF-I)-induced ERK and GSK-3β pathways; and 4) insulin, IGF-I or therapeutics (e.g., lithium) up-regulated the voltage-dependent Nav1.7 sodium channel.

Drug Development Targeting the Glycogen Synthase Kinase-3β (GSK-3β)-Mediated Signal Transduction Pathway: Targeting the Wnt Pathway and Transplantation Therapy as Strategies for Retinal Repair

Recent advances in stem cell biology have provided new insights that may lead to the development of regeneration therapy in the central nervous system to replenish lost neurons and to reconstitute neural circuits. The strategies for regeneration can be classified into two approaches: i) activation of endogenous neural stem cells and ii) transplantation of donor cells to replace lost cells. In the adult mammalian retina, Müller glia generate new retinal neurons in response to injury. The proliferation and differentiation of Müller glia–derived progenitors can be controlled by both intrinsic and extrinsic factors. Members of the Wnt/β-catenin signaling pathway, such as Wnt receptors and glycogen synthase kinase-3β, may be promising drug targets for neural regeneration. On the other hand, transplantation of photoreceptors or retinal pigment epithelia derived from human embryonic stem cells or induced pluripotent stem cells is also promising. Directed differentiation of pluripotent cells into retinal cells and purification to obtain retinal cells at a specific ontogenetic stage are required for donor cell preparation. Modulation of the host retinal environment to reduce the glial barrier is also critical for transplantation. To restore visual function, we need to understand the mechanisms underlying the integration of newly generated neurons or transplanted cells into the existing neural networks.

Drug Development Targeting the Glycogen Synthase Kinase-3β (GSK-3β)-Mediated Signal Transduction Pathway: Inhibitors of the Wnt/β-Catenin Signaling Pathway as Novel Anticancer Drugs

Accumulating evidence suggests that the Wnt/β-catenin signaling pathway is often involved in oncogenesis and cancer development. Accordingly, a novel anticancer drug can be developed using inhibitors of this pathway. However, at present, there is no selective inhibitor of this pathway available as a therapeutic agent. Although all the components of the Wnt/β-catenin signaling pathway can be a target for drug development, glycogen synthase kinase-3β (GSK-3β), in particular, may be a good target because GSK-3β is an essential component of the pathway, and activation of this kinase results in the inhibition of the Wnt signaling pathway. We found that the differentiation-inducing factors (DIFs), putative morphogens for Dictyostelium discoideum, inhibit the Wnt/β-catenin signaling pathway via the activation of GSK-3β, resulting in the cell-cycle arrest of human cancer cell lines. In this review, we summarize our recent findings on the antiproliferative effect of DIFs and show the possibility for development of a novel anticancer drug from DIFs and their derivatives.

Drug Development Targeting the Glycogen Synthase Kinase-3β (GSK-3β)-Mediated Signal Transduction Pathway: Role of GSK-3β in Adult Brain

Glycogen synthase kinase-3β (GSK-3β) is a constitutively active kinase. Since its activation results in neurofibrillary tangle (NFT) deposits in aged and Alzheimer’s disease (AD) brains, GSK-3β may be inhibited under normal conditions but activated under pathological conditions. Given its link to NFT formation, we sought to determine whether GSK-3β exists in the brain as a “pathological time bomb” that promotes disease development. To address this hypothesis, we analyzed GSK-3β heterozygote (GSK+/−) mice, which express GSK-3β at 50% wild-type levels. When tested in the Morris water maze test, GSK+/− mice surprisingly exhibited retrograde amnesia. Further analysis indicated that GSK+/− mice had impaired memory reconsolidation but normal memory consolidation. Therefore, we concluded that GSK-3β activation is required for memory reconsolidation in the adult brain.

Ion Channels Modulating Mouse Dendritic Cell Functions

Ca(2+)-mediated signal transduction pathways play a central regulatory role in dendritic cell (DC) responses to diverse Ags. However, the mechanisms leading to increased [Ca(2+)](i) upon DC activation remained ill-defined. In the present study, LPS treatment (100 ng/ml) of mouse DCs resulted in a rapid increase in [Ca(2+)](i), which was due to Ca(2+) release from intracellular stores and influx of extracellular Ca(2+) across the cell membrane. In whole-cell voltage-clamp experiments, LPS-induced currents exhibited properties similar to the currents through the Ca(2+) release-activated Ca(2+) channels (CRAC). These currents were highly selective for Ca(2+), exhibited a prominent inward rectification of the current-voltage relationship, and showed an anomalous mole fraction and a fast Ca(2+)-dependent inactivation. In addition, the LPS-induced increase of [Ca(2+)](i) was sensitive to margatoxin and ICAGEN-4, both inhibitors of voltage-gated K(+) (Kv) channels Kv1.3 and Kv1.5, respectively. MHC class II expression, CCL21-dependent migration, and TNF-alpha and IL-6 production decreased, whereas phagocytic capacity increased in LPS-stimulated DCs in the presence of both Kv channel inhibitors as well as the I(CRAC) inhibitor SKF-96365. Taken together, our results demonstrate that Ca(2+) influx in LPS-stimulated DCs occurs via Ca(2+) release-activated Ca(2+) channels, is sensitive to Kv channel activity, and is in turn critically important for DC maturation and functions.

PCR differential display-based identification of regulator of G protein signaling 10 as the target gene in human colon cancer cells induced by black tea polyphenol theaflavin monogallate

We have previously reported that black tea polyphenol theaflavin monogallate (TF-2) suppressed COX-2 and induced apoptosis in human colon cancer cells [Lu, J.B., Ho, C.-T., Ghai, G., Chen, K.Y., 2000. Differential effects of theaflavin monogallates on cell growth, apoptosis and Cox-2 gene expression in cancerous versus normal cells. Cancer Res. 60, 6465–6471.]. We now extended the study by using PCR-based differential display to search for genes that were selectively induced by TF-2. Here we report the identification of Regulator of G-binding protein signaling 10 (RGS10) as the target gene, which was induced as early as 4 h after the TF-2 treatment. We then examined the effect of TF-2 on several other RGS genes and found that, in addition to RGS10, TF-2 induced the expression of RGS14, but not RGS4. Other tea polyphenols, including theaflavin-3,3'-digallate (TF-3) and (−) epigallocatechin-3-gallate (EGCG), also induced the expression of RGS10 and RGS14, but not RGS4. However, theaflavin (TF-1), which does not contain the gallate moiety, was ineffective. These results showed for the first time that tea polyphenols can induce the expression of selective RGS genes and that the gallate moiety may be important in this induction. In view of the role of RGS in modulating G-protein mediated signal transduction pathways, our findings may be significant since dysregulation of G-signaling is prominently implicated in carcinogenesis.

Activated platelets stimulate migration of human granulosa cells via sphingosin1-phosphate

OBJECTIVE: The development of the corpus luteum (CL) is a complex process involving mechanisms of wound healing and tumor formation. Platelets are likely to be exuded into extravascular spaces among luteinizing granulosa cells and were reported to contain chemoattractive substances potentially capable of inducing endothelial migration, e.g. Sphingosin1-Phosphate (S1P). Thus, we investigated the effect of exudate activated platelets on migration of human granulosa cells. DESIGN: Laboratory study. MATERIALS AND METHODS: Study was performed with HGL5 cells, a human luteinized granulosa cell line, granulosa cells (HGC), were obtained from women undergoing IVF/ICSI treatment and supernatant of Thrombin activated platelets. Migration assays were performed with modified Boyden chambers and cell proliferation was determined with MTT. Platelet exudates (PE) were modified with heat, charcoal and alkaline phosphatase (AP). PE mediated signal transduction pathways were analyzed with specific inhibitors and phosphorylated antibodies for key molecules involved in cell migration.Each experiment was performed in triplicates and was repeated three times. The obtained data were analyzed by Student's t-test. RESULTS: Migration increased 2 fold at 20% PE in hGC and at 40% PE in HGL5 cells. PE induced migration was abolished by the pretreatment with Ly290004, an AKT inhibitor and PD98059, an ERK kinase inhibitor.We could show that PE induced migration leads to an activation of AKT and ERK.The heat treatment of PE only slightly reduced the migratory effect in both cell types; charcoal reduced the migration of hGC to 1.5 fold and to controls in HGL5 cells.The treatment with AP of PE decreased migration to untreated controls. The data indicate, that a heat stable and lipid platelet derived compound, which is biologically active after phosphorylation mediates the migratory effect of PE. In correlation to our previous data S1P, a bioactive lysophospholipid increased migration to 1.5 fold. CONCLUSIONS: This report demonstrates the induction of HGC migration, a key process of corpus luteum development is induced by PE. In addition, these effects were mediated at least through the phosphorylation of Akt and ERK Kinases. We have previously shown that Follicular High density lipoprotein associated lysophospholipid S1P is a novel mediator of human granulosa cell migration and we conclude from the obtained data, that activated platelets are another source of S1P, a novel mediator of corpus luteum development.

Biochemical and molecular analysis of LePS2;1: a phosphate starvation induced protein phosphatase gene from tomato

Adaptation of plants to phosphate (Pi) deficiency is a complex process involving host of biochemical changes. These changes are integrated at transcriptional level by Pi starvation mediated signal transduction pathway. Many of the signaling processes are regulated by reversible protein phosphorylation directed by protein kinases and protein phosphatases. In this study, we report the characterization of a protein phosphatase gene (LePS2;1) from tomato induced during phosphate starvation. The bacterially expressed recombinant LePS2;1 protein readily dephosphorylated a synthetic phospho-Ser/Thr peptide. Okadaic acid, an inhibitor of Ser/Thr protein phosphatases, suppressed the enzyme activity. Western blot analysis revealed the Pi starvation dependent accumulation of LePS2;1 protein. Over-expression of LePS2;1 in tomato plants resulted in increased anthocyanin accumulation and acid phosphatase activity under Pi sufficient condition. Transgenic plants exhibited distinct changes in morphology and delayed flower initiation. These results provide evidence that the protein phosphatase LePS2;1, plays an important role in phosphate starvation induced processes in tomato. To our knowledge this is the first comprehensive analysis of a protein phosphatase induced during phosphate starvation.

Xenopus V1R Vomeronasal Receptor Family Is Expressed in the Main Olfactory System

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.

Abrogation of U266 Multiple Myeloma Cell Proliferation Via Inhibition of NF-κB Activation by Curcumin

Background: Curcumin is a naturally occurring biologically active compound, and it has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. It is known for its anti-pro- liferative and proapoptotic effects in several cancer cells. Curcumin's effects on the mechanisms of cell survival and the expression of various cytokines were investigated in U266 cells and the in vivo effects of curcumin were examined using an animal model. Methods: Cell proliferation assay and flow cytometry were used to examine cell proliferation, along with cell cycle analysis. The protein expressions were analyzed by Western blotting and the expressed levels of cytokines were analyzed by the ELISA method. Results: Curcumin inhibited U266 cell growth in a dose-dependent and time-dependent manner. Cell cycle analysis showed an increased sub-G1 phase, a down regulated cyclinD1 expression and an induced p21 expression. Apoptosis induced a down regulated procaspase 3 expression and it induced cleaved PARP. Curcumin inhibited the IL (interleukin)-6 induced cell signal pathway via decreasing the STAT1 an 3, Erk cyclinD1 and c-myc expressions. Also, administration of 25mg/kg curcumin to a U266 animal model inhibited cancer cell engraftment in the bone marrow and it decreased the IL-6, sIL-6R and IL-8 ex- pression levels. Conclusion: Curcumin induced cell cycle arrest and apoptosis and it inhibited the IL-6 mediated signal transduction pathways in U266 cells. Similar to the in vitro results, curcumin inhibited cancer cell pro- liferation and the expression of cytokine in vivo. (Korean J Hematol 2008;43:19-27.)

Targeting the Sarcolemmal Calcium Pump: A Potential Novel Strategy for the Treatment of Cardiovascular Disease

Intracellular calcium dynamics play a very important role in mediating contraction and signalling in cardiomyocytes and vascular smooth muscle cells. A number of calcium transporters have been identified that orchestrate a complex process of excitation-contraction coupling and molecular signalling. Despite the variability of the calcium transporters expressed in cardiomyocytes, most calcium channel blockers used therapeutically target the L-type calcium channel and exhibit antihypertensive and/or vasodilating activities. Recently, another calcium pump which is located in the sarcolemma has been shown to mediate cardiac contractility and vascular tone. Interestingly, this sarcolemmal calcium pump (also known as Plasma Membrane Calcium/calmodulin dependent ATPase or PMCA) exerts its function not by altering global calcium concentration, but by mediating signal transduction pathways. This review will discuss recent advances that support the key roles of PMCA as signalling molecule and the potential to target this calcium pump as a novel approach for the treatment of cardiovascular disease.

Effects of development and dopamine depletion on striatal NMDA receptor-mediated calcium uptake

Calcium (Ca(2+)) is the currency of N-methyl-D-aspartate (NMDA) receptor mediated signal transduction pathways involved in the modification of synaptic efficacy during regulation of excitatory inputs into the striatum. The aim of the present study was to investigate the effects of development and dopamine depletion on NMDA receptor function. NMDA receptors were stimulated by incubation of striatal sections (350 microm) in buffer containing NMDA (100 microm) for 2 min, the slices were washed and uptake of radioactively labelled calcium ((45)Ca(2+)) was measured. Dopamine depletion has been reported to result in alterations of glutamate receptor expression and upregulation of NMDA receptor activity. However, the results of the present study show that dopamine depletion does not alter NMDA-stimulated Ca(2+) uptake into rat striatal slices in vitro. Unilateral striatal dopamine depletion was achieved by infusion of 6-hydroxydopamine (6-OHDA, 13.5 microg/4.5 microl) into the medial forebrain bundle (MFB) of the left hemisphere of ten rats. NMDA-stimulated (45)Ca(2+) uptake into striata following dopamine depletion was not significantly different from NMDA-stimulated (45)Ca(2+) uptake into striata obtained from sham-operated rats. Other factors that induce changes in NMDA receptor function include development and aging. In young rats aged 7 weeks old (n = 7) and 16 weeks old (n = 6) a significant 2-3 fold decrease in striatal NMDA receptor function was observed with increasing age over the 9 week period of development. To our knowledge these are the first results to show developmental decreases of NMDA receptor function in the striatum of juvenile rats.

The regulation of dendritic cell function by calcium-signaling and its inhibition by microbial pathogens

Dendritic cells (DC) are the sentinels of the immune system, linking innate with adaptive responses. The functional responses of DC are subject to complex regulation and serve as targets for pathogens. Ca2+-mediated signal transduction pathways serve a central regulatory role in DC responses to diverse antigens, including TLR ligands, intact bacteria, and microbial toxins. This review summarizes the major mechanisms of Ca2+-signaling that DC utilize to regulate maturation and antigen presentation, including a Ca2+-calmodulin (CaM)-CaM kinase II pathway that is localized to phagosomes and is targeted by the human intracellular pathogen, Mycobacterium tuberculosis. Restoration of functional Ca2+ signaling in DC may provide a novel mechanism to enhance therapy and promote vaccine efficacy to infectious diseases, including tuberculosis.

Phospholipase D-dependent and -independent p38MAPK activation pathways are required for superoxide production and chemotactic induction, respectively, in rat neutrophils stimulated by fMLP

Mitogen-activated protein kinase (MAPK)-mediated signal transduction pathways convert signals by extracellular stimulation into a variety of cellular functions. However, the roles of MAPKs in neutrophils are not well understood. To elucidate the temporal roles of p38MAPK during rat neutrophil activation stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), we examined the kinetics of this enzyme and the role of p38MAPK related to neutrophil functions (superoxide production and chemotaxis). SB203580, a potent and specific inhibitor of p38MAPK, significantly depressed both superoxide production and chemotaxis. Ethanol and 1-butanol, inhibitors of phospholipase D (PLD), suppressed p38MAPK activation in neutrophils under conditions (1 μM fMLP for 5 min) that stimulated superoxide production; and they significantly depressed superoxide production in rat neutrophils stimulated by fMLP. However, neither inhibitor had any effect on the activation of p38MAPK under the conditions (10 nM fMLP for 60 min) that gave optimal chemotaxis. These results indicate that multiple signaling pathways were involved in stimulating p38MAPK and that p38MAPK played different roles in regulating neutrophil function depending on the conditions for stimulation with fMLP. In addition, the activation of p38MAPK occurred dependent on or independent of PLD activation in neutrophils stimulated with fMLP.

Clinical Pharmacodynamic Effects of the Growth Hormone Receptor Antagonist Pegvisomant: Implications for Cancer Therapy

The present study evaluated and compared the efficacy of pegvisomant and octreotide in blocking the growth hormone (GH) axis in humans based on pharmacodynamic biomarkers associated with the GH axis. The study also evaluated the safety of pegvisomant given at high s.c. doses for 14 days. Eighty healthy subjects were enrolled in five cohorts: cohorts 1 to 3, s.c. pegvisomant at 40, 60, or 80 mg once dailyx14 days (n=18 per cohort); cohort 4, s.c. octreotide at 200 microg thrice dailyx14 days (n=18); and cohort 5, untreated control (n=8). Serial blood samples were collected to measure plasma concentrations of total insulin-like growth factor type I (IGF-I), free IGF-I, IGF-II, IGF-binding protein 3 (IGFBP-3), and GH in all subjects and serum pegvisomant concentrations in subjects of cohorts 1 to 3. All subjects receiving treatment were monitored for adverse events (AE). After s.c. dosing of pegvisomant once daily for 14 days, the mean maximum suppression values of total IGF-I were 57%, 60%, and 62%, at 40, 60, and 80 mg dose levels, respectively. The maximum suppression was achieved approximately 7 days after the last dose and was sustained for approximately 21 days. Pegvisomant also led to a sustained reduction in free IGF-I, IGFBP-3, and IGF-II concentrations by up to 33%, 46%, and 35%, respectively, and an increase in GH levels. In comparison, octreotide resulted in a considerably weaker inhibition of total IGF-I and IGFBP-3 for a much shorter duration, and no inhibition of IGF-II. AEs in pegvisomant-treated subjects were generally either grade 1 or 2. The most frequent treatment-related AEs included injection site reactions, headache, and fatigue. Pegvisomant at well-tolerated s.c. doses was considerably more efficacious than octreotide in suppressing the GH axis, resulting in substantial and sustained inhibition of circulating IGF-I, IGF-II, and IGFBP-3 concentrations. These results provide evidence in favor of further testing the hypothesis that pegvisomant, through blocking the GH receptor-mediated signal transduction pathways, could be effective in treating tumors that may be GH, IGF-I, and/or IGF-II dependent, such as breast and colorectal cancer.

Effect of Simulated Microgravity on Receptor Mediated Signal Transduction in Hind Limb Suspended Animal Model

Microgravity is an extreme environment that affects cellular processes. However, changes in receptor mediated signal transduction processes under such an environment have not been well understood. In this study, we have used a NASA validated Hind Limb Suspension (HLS) model to induce weightlessness in rats and mice and examined any changes in molecules that constitute receptor mediated signal transduction pathways. Animals were hind limb suspended by tail for 21 days. The body weight, food and water intake was monitored. At term, tissue samples were harvested in 20 mM PBS, pH 7.4 and processed for biochemical analyses. Western blots using specific antibodies to alpha subunits of stimulatory (Gs), inhibitory (Gi-1/2) and Gq, a G-protein that couples activation of phosphatidylinositol specific phospholipase C to produce inositol (1,4,5) trisphosphate, showed an increased amounts of Gs, Gi and Gq -proteins in lung, liver, kidney and heart samples of HLS animals as compared to controls. Further, Gq mediated production of inositol phosphates was measured following labeling of rat lung tissues with 3[H]-myo-inositol and analysis by anion exchange chromatography. Results suggested an increased accumulation of total inositol phosphates in HLS animals than in controls. In conclusion, microgravity may affect signaling molecules that mediate cellular signal transduction processes. (Supported by Arkansas Space Grant Consortium, Grant No. UALR-14210).

A molecular dynamics analysis of the GCC-box binding domain in ethylene-responsive element binding factors

Ethylene-responsive element (ERE) binding factors is responsible for a consensus nucleotide sequence AGCCGCC (GCC-box) binding in many important process of plant growing through gene regulation and mediating signal transduction pathways in response to environmental stress. The GCC-box binding domain (GBD) as a novel fold for DNA recognition has been analyzed by means of molecular dynamics. The simulations show that the complex of GBD–DNA trajectories show similar fluctuations in the atomic positions as uncomplexed, particularly at three β strands involving DNA binding. The calculations of entropy also affirm that GBD flexibility is basically similar for two ligation states. Further, the two complexation states present similar patterns of concerted motions, indicating that the bound DNA cannot alter GBD flexibility. It is inferred that the flexibility of GBD molecule is independent of its ligation state. So in the protein–DNA recognition, the GBD cannot be easily induced while DNA shows better flexibility. Comparison between simulations of unligated GBD and the complexed GBD (in isolation or DNA-bound) reveals intrinsic flexibilities in some certain parts of the molecule play a key role in DNA recognition. In addition, MD simulation identifies that water molecule may mediate interaction between GBD and DNA.

Protein kinase Cα and sphingosine 1-phosphate-dependent signaling in endothelial cell

Protein kinase C (PKC)-mediated signal transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the role of various PKC isoforms in sphingosine 1-phosphate (S1P)-stimulated endothelial cells is not well understood. PKCα and PKCɛ activity are increased in endothelial cell cultures, and S1P receptor transfection studies indicate S1P 3 stimulates PKCα and S1P1 leads to PKCɛ activity. Infection of endothelial cells with dominant negative (DN)PKCα adenovirus reduces cell migration and greatly inhibits morphogenesis in cells stimulated with S1P. This effect is specific to PKCα, as infection with DN PKCɛ does not alter either migration or morphogenesis. The PKC-specific chemical inhibitor GF109203X also inhibits these two responses. Infection of endothelial cells with dominant negative PKCα reduces S1P-induced calcium rise. This maximal rise requires calcium uptake, but it does not require enzymatic activity of the kinase. Pretreatment of these cells with the PKC-specific inhibitor GF109203X does not inhibit S1P-induced calcium rise. S1P-induced morphogenesis but not cell migration is critically dependent on extracellular calcium. Pretreatment of endothelial cells with phorbol 12-myristate 13-acetate for 5 min abolishes S1P-stimulated rise in calcium but had little or no effect on migration. The PMA-inhibited calcium rise can be prevented by PKC inhibitor or infection with dominant negative PKCα.

Glutathione Reductase in Leaves of Cowpea: Cloning of Two cDNAs, Expression and Enzymatic Activity under Progressive Drought Stress, Desiccation and Abscisic Acid Treatment

Reactive oxygen species are frequently produced when plants are exposed to abiotic stresses. Among the detoxication systems, two enzymes, ascorbate peroxidase and glutathione reductase (GR) play key roles. GR has also a central role in keeping the reduced glutathione pool during stress thus allowing the adjustments on the cellular redox reactions. The aim of this work was to study the variations in cytosolic and dual-targeted GR gene expression in the leaves of cowpea plants submitted to progressive drought, rapid desiccation and application of exogenous abscisic acid (ABA). Two cowpea (Vigna unguiculata) cultivars, one drought-resistant ('EPACE-1'), the other drought-sensitive ('1183') were submitted to progressive drought stress by withholding irrigation. Cut-off leaves were air-dried or treated with exogenous ABA. Two GR cDNAs, one cytosolic, the other dual-targeted to chloroplasts and mitochondria were isolated by PCR and cloned in plasmid vectors. Reverse-transcription PCR was used to study the variations in GR gene expression. Two new cDNAs encoding a putative dual-targeted and a cytosolic GR were cloned and sequenced from leaves of V. unguiculata. Drought stress induced an up-regulation of the expression of the cytosolic GR gene directly related to the intensity of the stress in both cultivars. The expression of dual-targeted GR was up-regulated by the drought treatment in the susceptible cultivar only. Under a fast desiccation, the '1183' cultivar responded later than the 'EPACE-1', although in 'EPACE-1' it was the cytosolic isoform which responded and in '1183' the dual-targeted one. Exogenous ABA enhanced significantly the activity and expression levels of GR in both cultivars after treatment for 24 h. These results demonstrate a noticeable activation in both cultivars of the antioxidant metabolism under a progressive water stress, which involves both GR genes in the case of the susceptible cultivar. Under a fast desiccation, the susceptible cultivar responded later than the resistant one, suggesting a weaker capacity of response versus the resistant one. Exogenous ABA probably acts on GR gene expression via a mediated signal transduction pathway.

Paxillin interacts with bcl‐2 through its LD4 domain

Paxillin is an adapter protein implicated in growth factor and integrin mediated signal transduction pathways. It is the founding member of a family of proteins that include Hic-5 and leupaxin, which share many of paxillin’s structural characteristics. Paxillin can function during the regulation of cell migration and spreading. Loss of paxillin results in inhibition of focal adhesion turnover, loss of polarization and loss of directional motility. However, its role during organogenesis requires further delineation. Paxillin-deficient mice are abnormal by embryonic day 9.5, suggesting that paxillin may play an important role during development. Previous work from this laboratory demonstrated that paxillin interacted with bcl-2’s BH4 domain influencing branching morphogenesis in the embryonic kidney. Here studies were performed to determine the bcl-2 binding motif in paxillin. Bcl-2 was found to associate with the amino-terminal portion of paxillin, specifically the LD4 motif. This interaction was found to be specific for paxillin since Hic-5 and leupaxin LD4 motifs did not associate with bcl-2. Furthermore, incubation of embryonic kidneys with the LD4 domain had a profound effect on ureteric bud branching and morphogenesis. These data suggest that bcl-2 association with paxillin plays a unique role during kidney development that paxillin’s other family members may not be able to fulfill.