1321N1 Cells Research Articles

Immobilized P2X 2 purinergic receptor stationary phase for chromatographic determination of pharmacological properties and drug screening

The purinergic receptor signaling system plays an important role in communication between cells in the nervous system and opens new opportunities for screening of potential drugs. Our objective was to explore the pharmacological properties and establish a new methodology for ligand screening for the P2X 2 receptor, which has been developed by the combinatorial library approach Systematic Evolution of Ligands by Exponential enrichment (SELEX). To this end, membranes of 1321N1 cells stably transfected with rat P2X 2 receptors were resuspended in 2% cholate detergent and subsequently coupled onto an immobilized artificial membrane (IAM). The IAM–cholate–P2X 2 mixture was then dialyzed, centrifuged and packed into a FPLC column. Equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2′3′- O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using 32P alpha ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, confirming previous studies [C.A. Trujillo, A.A. Nery, A.H. Martins, P. Majumder, F.A. Gonzalez, H. Ulrich, Biochemistry 45 (2006) 224–233]. In addition, we demonstrate that this assay can be used in in vitro selection procedures for RNA aptamers binding to P2X 2 receptors. The results demonstrate that the receptor can be immobilized in a stable format and reused over an extended period of time, facilitating the exploration of ligand–receptor interactions and screening of combinatorial pools for possible ligands.

The synthesis and initial characterization of an immobilized purinergic receptor (P2Y 1) liquid chromatography stationary phase for online screening

The synthesis and initial characterization of an immobilized purinergic receptor (P2Y 1) liquid chromatography stationary phase for online screening

Cyathane diterpenes from Sarcodon cyrneus and evaluation of their activities of neuritegenesis and nerve growth factor production

Two new cyathane diterpenes, cyrneine C ( 4) and D ( 5), were isolated from the mushroom Sarcodon cyrneus, along with previously isolated cyrneine A, B and glaucopine C. The structures of the novel diterpenoids were determined by the analysis of spectroscopic data. Effects of the cyrneines and glaucopine C on the NGF gene expression in 1321N1 cells and on neurite outgrowth on PC12 cells were evaluated.

Sodium valproate at therapeutic concentrations changes Ca 2+response accompanied with its weak inhibition of protein kinase C in human astrocytoma cells

Sodium valproate (VPA) has been used clinically for treatment of not only epilepsy but also mood disorder. Although VPA is effective for treatment of epilepsy via inhibition of γ-aminobutyric acid transaminase, it remains unknown why VPA is effective for the treatment of mood disorder. The authors examined the effect of VPA at therapeutic concentrations (300 and 600 μM) on the elevation of intracellular free calcium concentration ([Ca 2+] i) induced by carbachol, a muscarinic receptor agonist, in 1321N1 human astrocytoma cells. Treatment of the cells with 300 and 600 μM VPA for 2 min did not change the carbachol-induced [Ca 2+] i elevation. Treatment with 300 and 600 μM VPA for 48 h, however, reduced the elevation. Since we have shown that Li + reduced carbachol-induced [Ca 2+] i elevation in protein kinase C (PKC)-downregulated 1321N1 cells [Kurita, M., Mashiko, H., Rai, M., Kumasaka, T., Kouno, S., Niwa, S., Nakahata, N., 2002. Lithium chloride at a therapeutic concentration reduces Ca 2+response in protein kinase C down-regulated human astrocytoma cells, Eur. J. Pharmacol. 442, 17–22.], the activity of PKC was examined. Treatment with VPA at the same concentrations for 24 or 48 h weakly reduced protein kinase C activity in membrane and cytosol fractions from the cells. On the other hand, the treatment of the cells with 600 μM VPA for 24 or 48 h slightly increased the B max value, but not the K d value, in the binding of [ 3H]quinuclidinyl benzylate, a muscarinic receptor ligand, to the membranes, suggesting that the number or affinity of muscarinic receptor did not decrease after VPA treatment. These results indicate that VPA at therapeutic concentrations slightly decreases the PKC activity and inhibits muscarinic receptor-mediated [Ca 2+] i elevation probably through change in the intracellular signaling pathway. VPA-induced reduction of PKC activity and [Ca 2+] i elevation may play a role in the treatment of mood disorder.

Alteration of Adenylyl Cyclase Type 6 Expression in Human Astrocytoma Cells After Exposure to Simulated Microgravity

Although many physiological changes after space flight have been reported, it is not clear how microgravity influences our bodies. The focus of the present study was to clarify the changes in G-protein-coupled receptor-mediated intracellular signaling, especially Gs-adenylyl cyclase (AC)-adenosine 3 � ,5 � -cyclic monophosphate (cyclic AMP) pathway, under simulated microgravity. Human astrocytoma 1321N1 cells were cultivated under vector-averaged microgravity conditions generated by clinostat rotation (20 rpm) for 24 hr. Isoproterenol, a β-adrenergic agonist and forskolin, a direct AC stimulant, increased intracellular cyclic AMP level in concentration dependent manners, however, both of which response were decreased in cells cultivated in clinostat rotation. While the level of Gαs or intracellular ATP, a substrate for AC, was not changed, the AC activity was significantly low in the membranes of clinostat-rotated cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that AC type 3 (AC3), AC6, and AC9 and to a lesser extent AC7 and AC8 were expressed in 1321N1 cells. Among them, the expression of AC6 mRNA was significantly decreased by clinosta tr otation. These results indicate that intracellular cyclic AMP production by agonists may be decreased via a reduction in AC6 expression under simulated microgravity conditions.

The use of monoreassortants and reverse genetics to map reovirus lysis of a ras-transformed cell line

Reovirus has been shown to lyse most transformed cells while establishing a persistent or abortive infection in non-transformed cells. Developing methods to identify the reovirus genes associated with oncolysis is an important step toward understanding the mechanisms involved. This report is the first to develop and apply the use of monoreassortants and reverse genetics to identify an individual reovirus gene associated with reovirus oncolysis. Infection with reovirus serotypes 1/Lang, 2/Jones or 3/Dearing of cells transformed with a normal copy of c-Ha-RAS (N1 cells) or with a normal copy of c-Myc (Myc-3 cells), produces large amounts of progeny virus of all three serotypes and results in lysis of both these cell lines. Infection of cells transformed with a mutant c-Ha-RAS gene (T1 cells) with either serotype 1/Lang and 2/Jones results in the production of large amounts of virus and lysis of the cells. In sharp contrast, serotype 3/Dearing virus infection of these cells produced small amounts of virus and resulted in limited lysis of these cells. Using monoreassortants and reverse genetics we exploited this phenotypic difference between the three serotypes to identify a single reovirus gene linked to the preferential lysis of the T1 cells, the S4 gene.

Calmodulin potentiates Gβγ activation of phospholipase C-β3

Phospholipase C-β (PLC-β) isozymes (EC 3.1.4.11) hydrolyze the membrane phospholipid phosphatidylinositol-4,5-bisphosphate to generate intracellular second messenger signaling molecules inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation and other cellular stimuli. PLCβ1 and PLCβ3 isozymes were previously demonstrated to bind the calcium-sensitive molecule calmodulin [McCullar JS, Larsen SA, Millimaki RA, Filtz TM. Calmodulin is a phospholipase C-{beta} interacting protein. J Biol Chem 2003;278(36):33708–13]. We have now shown through fluorescence anisotropy that calmodulin/PLCβ3 affinities increase with increasing calcium in a physiologically relevant concentration range. The bimolecular affinity constants for calmodulin interaction with PLCβ1 or PLCβ3 were estimated as 260 and 200nM, respectively, from fluorescence anisotropy data. There was no effect of calmodulin on basal or Gαq-stimulated catalytic activity for either isozyme. However, the interaction between calmodulin and PLCβ3 leads to potentiation of activation by the G-protein βγ dimer in an in vitro assay. 1321N1 cells treated with calmodulin inhibitors concurrent with and post-stimulation of muscarinic receptors significantly reduced [3H]PIP hydrolysis. Together these data are suggestive of cooperative role for calmodulin in the G-protein βγ dimer-stimulated activity of PLCβ3.

Extracellular NAD+ Is an Agonist of the Human P2Y11 Purinergic Receptor in Human Granulocytes

Extracellular NAD+ Is an Agonist of the Human P2Y11 Purinergic Receptor in Human Granulocytes

Opposite diastereoselective activation of P2Y1 and P2Y11 nucleotide receptors by adenosine 5′‐O‐(α‐boranotriphosphate) analogues

We explored the stereoselective activation of the P2Y11 receptor, stably expressed and tagged with GFP, in 1321N1 cells, in comparison to its closest homologue, the P2Y1 receptor. The potency of several chiral ATP analogues was determined by measuring increases in intracellular calcium concentration ([Ca2+]i). In a series of ATP-alpha-B and ATP-alpha-S analogues, a non-bridging oxygen atom of Palpha was substituted by BH3 or sulphur, respectively, introducing a chiral center at Palpha. The pairs of diastereoisomers (A and B isomers) were each applied as purified compounds. The (B) isomers (ATP-alpha-B Sp isomers and ATP-alpha-S Rp isomers) of all derivatives tested were more potent at the P2Y11 receptor than the corresponding (A) isomers (ATP-alpha-B Rp isomers and ATP-alpha-S Sp isomers) and the parent compounds. This characteristic of the P2Y11 receptor is opposite to the behaviour of the same diastereoisomers at the P2Y1 receptor, at which the (A) isomers are more active. The distinctly opposite diastereoselective activity of ATP derivatives at the P2Y11 and the P2Y1 receptor will allow the deciphering of structural differences of the ligand recognition sites between these receptor subtypes and may aid in the development of subtype-selective agonists. Moreover, ATP-alpha-B diastereoisomers are not active at the P2Y2 receptor. Thus, they are compounds suitable for distinguishing the functional contribution of the two ATP-activated P2Y receptors, the P2Y2 and P2Y11 receptor, in physiological or pathophysiological responses of cells.

Extracellular NAD+Is an Agonist of the Human P2Y11Purinergic Receptor in Human Granulocytes

Micromolar concentrations of extracellular beta-NAD+ (NAD(e)+) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)+ generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)+-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.

Distinct Roles for Protein Kinase C Isoforms in Regulating Platelet Purinergic Receptor Function

ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking.

Expression of endothelins and their receptors in glioblastoma cell lines

The endothelins (ETs) are a family of three peptides named ET-1, ET-2 and ET-3 that have been initially isolated as potent vasoactive peptides; ETs are synthesized as precursor proteins (preproETs) and are activated by proteolytic cleavage. ETs exert their biological effects through the activation of two receptors subtypes, ETA and ETB. Recent studies have shown that, besides its vascular effects, ET-1 appears to play a major role also in the growth and progression of various types of cancers and ETA or ETB are alternatively indicated as mediators of the ET-1 biological effects. In this study we have investigated the expression and the amounts of preproET-1, preproET-2, ETA and ETB receptors mRNA by classical RT-PCR and quantitative real-time PCR in one human low grade astrocytoma cell line and eight human glioblastoma cell lines. PCR products corresponding to ETB receptor and preproET-1 were detectable in all the cell lines whilst ETA receptor and preproET-2 were only detected in five cell lines. Quantitative real-time PCR experiments showed wide differences in the amounts of mRNAs among the cell lines examined. Range values were 0.23-4860.51 fg/mug total cDNA for preproET-1; 0.13-3330.18 fg/mug total cDNA for preproET-2; 0.63-286.12 fg/mug total cDNA for ETA and 14.40-6720.67 fg/mug total cDNA for ETB. We measured the ET-1 released in the extracellular medium by an ELISA assay and we found an excellent correlation (correlation coefficient r = 0.9526, P = 0.0003) between the amounts of preproET-1 mRNA and released ET-1 peptide. Finally, in the 1321N1 cell line ETB receptors are functionally coupled to intracellular signaling pathways because the stimulation of ETB receptors by ET-1 induces the phosphorylation of the extracellular regulated kinases (ERKs). Although the majority of glioblastoma cell lines in culture express ET isoforms and ET receptors, we conclude that ET-1 and the ETB receptors are likely to mediate the effects of the ET system in glioblastoma cell lines.

Modulation of human TRPV1 receptor activity by extracellular protons and host cell expression system

The transient receptor potential vanilloid 1 (TRPV1) receptor is a ligand-gated cation channel that can be activated by capsaicin, heat, protons and cytosolic lipids. We compared activation of recombinant human TRPV1 receptors stably expressed in human 293 cells, derived from kidney embryonic cells, and in human 1321N1 cells, derived from brain astrocytes. Cellular influx of calcium was measured in response to acid, endovanilloids ( N-arachidonoyl-dopamine, N-oleoyl-dopamine and anandamide), capsaicin and other traditional vanilloid agonists under normal (pH 7.4) and acidic (pH 6.7 and 6.0) assay conditions. The host cell expression system altered the agonist profile of endogenous TRPV1 receptor agonists without affecting the pharmacological profile of either exogenous TRPV1 receptor agonists or antagonists. Our data signify that the host cell expression system plays a modulatory role in TRPV1 receptor activity, and suggests that activation of native human TRPV1 receptors in vivo will be dependent on cell-specific regulatory factors/pathways.

P2Y2 receptor polymorphisms and haplotypes in cystic fibrosis and their impact on Ca2+ influx

Activation of P2Y2 receptors in airway epithelia by ATP and UTP stimulates a Ca2+-regulated Cl- channel, which regulates Cl- secretion in cystic fibrosis (CF). We hypothesized that genetic alterations in the P2Y2 receptor may act as disease modifiers in CF and thus analyzed the coding region of this gene for polymorphisms in 146 CF patients and 64 healthy controls. We also assessed the impact of the genetic variants on Ca2+-influx of P2Y2-null cells transfected with several P2Y2 receptor haplotypes. We identified three frequent nonsynonymous P2Y2 receptor polymorphisms: Leu46Pro; Arg312Ser and Arg334Cys, of which only Arg312Ser was significantly more common in CF: Arg = 0.80, Ser = 0.20 (CF) vs. Arg = 0.72, Ser = 0.28 (controls), P < 0.05; for Leu46Pro, Leu = 0.92, Pro = 0.08 (CF) vs. Leu = 0.96, Pro = 0.04 (controls), P = 0.65 and for Arg334Cys, Arg = 0.79, Cys = 0.21 (CF) vs. Arg = 0.84, Cys = 0.16 (controls), P = 0.79. The most frequent haplotype was Leu46Leu/Arg312Arg/Arg334Arg (28% in CF, 31% in controls) but 6% of CF patients and none of the controls had Leu46Leu/Ser312Ser/Arg334Cys or Leu46Leu/Arg312Arg/Cys334Cys. To assess function of the receptor haplotypes, we stably transfected 1321N1 (P2Y-null) cells to similar levels of mRNA expression with Leu46Leu/Arg312Arg/Arg334Arg (wild-type), Leu46Leu/Ser312Ser/Arg334Arg and Leu46Leu/Arg312Arg/Cys334Cys and measured ATP-stimulated transient Ca2+-influx. Cells expressing the homozygous Cys334 variant had significantly increased Ca2+-influx compared to wild-type (P<0.01). The increase in Ca2+-influx was more pronounced in cells carrying the homozygous Ser312 variant than in cells with the other two genotypes (P<0.01). These data indicate that P2Y2 receptor gene haplotypes influence intracellular Ca2+-release. Such genetic variants might therefore represent modifiers of Cl- secretion or of response to P2Y2 agonist therapy in CF.

Cloning and Characterization of the Porcine P2Y6 Receptor: Evidence for Gi Protein‐mediated Signaling in Coronary Smooth Muscle

The P2Y6 receptor (P2Y6R) has been thought exclusively coupled with Gq proteins. Our previous work has shown that P2Y6R mRNA is highly expressed in porcine coronary smooth muscles. However, the molecular and pharmacological properties and the functional role of this receptor remain undefined. Here we report the cloning of the cDNA for this receptor that encodes an open reading frame for a protein of 328 amino acids with highest homology to the human P2Y6R. Heterologous expression of this receptor in 1321N1 cells reveals that UDP was more potent and efficacious than UTP for increasing intracellular [Ca2+]i, and ATP/ADP were inactive. However, homologous over-expression of the P2Y6R in sub-cultured coronary smooth muscle cells did not increase intracellular [Ca2+]i in response to UDP, but it enabled UDP to suppress forskolin-induced cAMP elevation, which was eliminated by PTX pretreatment, indicating Gi protein-mediated signaling in coronary smooth muscles. Consistent with this interpretation, UDP stimulation of intact coronary arteries denuded of endothelium decreased isoproterenol-induced vessel relaxation and cAMP elevation. In addition, UDP increased total DNA and protein synthesis in sub-cultured coronary smooth muscle cells over-expressing P2Y6R and in intact coronary arteries, both of which were prevented by PTX. In conclusion, we have cloned and characterized the porcine P2Y6R and demonstrated that this receptor is primarily coupled with Gi protein in coronary smooth muscles to mediate UDP-induced cell proliferation and inhibition of vasorelaxation. (Support: HL29582 to PED and HL062552 to MS)

Role of S1P signaling in TNF‐mediated 1321N1 cell proliferation

TNF activates sphingosine kinase (SphK), resulting in the formation of sphingosine 1 phosphate (S1P). S1P is a ligand for its cognate cell-surface GPCRs, however S1P can also act intracellularly as a second messenger. In this study, we examined the role of S1P signaling in mediating the proliferative effects of TNF on 1321N1 astrocytoma cells. Inhibition of SphK with either of two pharmacologic inhibitors attenuated TNF-stimulated DNA synthesis without affecting the response to serum. Thus, S1P formation appears to be necessary for TNF-stimulated DNA synthesis. The signal transduction pathway for TNF-stimulated DNA synthesis was then characterized by pharmacologic inhibition. TNF-stimulated DNA synthesis was found to require Rho, Rho kinase and ERK. However, Rho and ERK activation by TNF are not mediated through SphK, as the SphK inhibitors do not block these responses. In contrast, inhibition of PI3K or Akt attenuated TNF-induced DNA synthesis and the ability of TNF to induce Akt phosphorylation was attenuated by SphK inhibitors. These data suggest that Akt is the downstream effector of the SphK-dependent effects of TNF. TNF was also found to attenuate expression of p27, a cell cycle inhibitor known to be regulated by Akt. Current studies are focused on whether p27 downregulation is mediated through SphK and whether this is the mechanism by which SphK affects cell cycle regulation. Additionally, data with PTX suggests the actions of SphK and S1P induced by TNF are not via cell surface GPCRs, thus the mechanism of how S1P affects Akt is under study.

Neurotrophic factor production in human astrocytoma cells by 2,5,6-tribromogramine via activation of epsilon isoform of protein kinase C

It is known that astrocytes secrete several neurotrophic factors to promote the survival of neurons. For the treatment of neuronal disorders, low molecular weight compounds inducing neurotrophic factor synthesis are useful, because neurotrophic factors are polypeptides which cannot cross the blood brain barrier. When rat pheochromocytoma (PC-12) cells were cultivated in the medium of human astrocytoma cells (1321N1) treated with 2,5,6-tribromogramine, they differentiated to neuron-like cells possessing neurites, indicating that 2,5,6-tribromogramine released neurotrophic factors from 1321N1 cells. In fact, 2,5,6-tribromogramine increased nerve growth factor (NGF) protein synthesis and secretion through mRNA expression. 2,5,6-Tribromogramine inhibited carbachol-induced phosphoinositide hydrolysis as well as phorbol 12,13-myristate acetate did. The inhibition was recovered by bisindolylmaleimide I (GF109203X), a specific protein kinase C (PKC) inhibitor, indicating that 2,5,6-tribromogramine may activate PKC. The morphological differentiation of PC-12 cells by the medium treated with 2,5,6-tribromogramine was also reduced by GF109203X. 2,5,6-Tribromogramine translocated PKC-ɛ but not PKC-α or PKC-ζ, to membrane fraction from cytosol fraction. These results indicate that 2,5,6-tribromogramine promotes the synthesis and secretion of neurotrophic factors including NGF in 1321N1 cells via an activation of PKC-ɛ.

A Poisson limit theorem for a two-stage equiprobable scheme of allocating particles into cells

We consider a two-stage scheme of allocating particles into cells. At the first stage, N0 initial particles are independently and equiprobably allocated into N1 cells of the first layer. At the second stage, these N1 cells are taken as particles which are independently and equiprobably allocated into N2 cells of the second layer. We present conditions under which the distribution of the number of cells of the second layer containing exactly r particles converges to the Poisson law.

Transactivation of Platelet-Derived Growth Factor Receptor α by the GTPase-Deficient Activated Mutant of Gα12

The GTPase-deficient, activated mutant of Galpha12 (Galpha12Q229L, or Galpha12QL) induces neoplastic growth and oncogenic transformation of NIH 3T3 cells. Using microarray analysis, we have previously identified a role for platelet-derived growth factor receptor alpha (PDGFRalpha) in Galpha12-mediated cell growth (R. N. Kumar et al., Cell Biochem. Biophys. 41:63-73, 2004). In the present study, we report that Galpha12QL stimulates the functional expression of PDGFRalpha and demonstrate that the expression of PDGFRalpha by Galpha12QL is dependent on the small GTPase Rho. Our results indicate that it is cell type independent as the transient expression of Galpha12QL or the activation of Galpha12-coupled receptors stimulates the expression of PDGFRalpha in NIH 3T3 as well as in human astrocytoma 1321N1 cells. Furthermore, we demonstrate the presence of an autocrine loop involving PDGF-A and PDGFRalpha in Galpha12QL-transformed cells. Analysis of the functional consequences of the Galpha12-PDGFRalpha signaling axis indicates that Galpha12 stimulates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through PDGFR. In addition, we show that Galpha12QL stimulates the phosphorylation of forkhead transcription factor FKHRL1 via AKT in a PDGFRalpha- and PI3K-dependent manner. Since AKT promotes cell growth by blocking the transcription of antiproliferative genes through the inhibitory phosphorylation of forkhead transcription factors, our results describe for the first time a PDGFRalpha-dependent signaling pathway involving PI3K-AKT-FKHRL1, regulated by Galpha12QL in promoting cell growth. Consistent with this view, we demonstrate that the expression of a dominant negative mutant of PDGFRalpha attenuated Galpha12-mediated neoplastic transformation of NIH 3T3 cells.

Activity of recombinant trypsin isoforms on human proteinase‐activated receptors (PAR): mesotrypsin cannot activate epithelial PAR‐1, ‐2, but weakly activates brain PAR‐1

Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs. Using fura 2 [Ca(2+)](i) measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca(2+) response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-1 levels (<11%), half-maximal responses were seen for both cationic and anionic trypsins at about 5 nM. In the epithelial cells, mesotrypsin did not activate PAR-2 or PAR-1, whereas both anionic and cationic trypsins were comparable activators. We also investigated human astrocytoma 1321N1cells, which express PAR-1 and some PAR-3, but no PAR-2. High concentrations (>100 nM) of mesotrypsin produced a relatively weak Ca(2+) signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nM mesotrypsin, and at 10-20 nM cationic and anionic trypsins. Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca(2+)](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.