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BBA - Molecular Cell Research (v.1763, #4)

Editorial Board (pp. ii).

Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells by Chisato Kosugi-Tanaka; Xuefei Li; Chenjuan Yao; Tetsuya Akamatsu; Norio Kanamori; Kazuo Hosoi (pp. 337-344).

The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP–AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5–GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP–AQP5–T259A and AQP5–GFP–T259A. They were used to transfect Madin–Darby canine kidney (MDCK) cells. The GFP–AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N⁶, 2′- O-dibutyryladenosine 3′, 5′-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-( p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl-dl-carnitine chloride (PCC). In contrast, the AQP5–GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP–AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5–GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.

Keywords: Abbreviations; AQP5; aquaporin 5; dbcAMP,N; ⁶; 2′-; O; -dibutyryladenosine 3′, 5′-cyclic monophosphate; DMEM/F-12; Dulbecco's modified Eagle's medium F-12 HAM; ECL; enhanced chemiluminescence; EDTA; ethylenediaminetetraacetic acid; FBS; fetal bovine serum; GFP; green fluorescent protein; GLUT; glucose transporter; HRP; horseradish peroxidase; H-89; N-[2-(; p; -bromocinnamylamino)ethyl]-5-isoquinolinesulfoamide; MDCK; Madin–Darby canine kidney; PBS; phosphate-buffered saline; PCC; palmitoyl-; dl; -carnitine chloride; PKA; protein kinase A; PKC; protein kinase C; PKG; protein kinase G; PMA; phorbol 12-myristate 13-acetate; PMSF; phenyl methylsulfonyl fluoride; SDS-PAGE; sodium dodecylsulfate-polyacrylamide gel electrophoresisAquaporin 5; Trafficking; GFP-tagged AQP5

Systematic search for the rate constants that control the exocytotic process from chromaffin cells by a Genetic Algorithm by Aviv Mezer; Uri Ashery; Menachem Gutman; Elad Project; Eran Bosis; Gadi Fibich; Esther Nachliel (pp. 345-355).

We have recently created a kinetic model that reproduces the dynamics of exocytosis with high accuracy. The reconstruction necessitated a search, in a multi-dimensional parameter space, for 37 parameters that described the system, with no assurance that the parameters, which reconstructed the observations, are a unique set. In the present study, a Genetic Algorithm (GA) was used for a thorough search in the unknown parameter space, using a strategy of gradual increase of the complexity of the analyzed input data. Upon systematic incorporation of one to four measurable parameters, used as input signals for the analysis, the constraint set on the GA search imposed the convergence of the free parameters into a single narrow range. The mean values for each adjustable parameter represent a minimum for the fitness function in the multi-dimensional parameter space. The GA search demonstrates that the parameters that control the kinetics of exocytosis are the rate constants of the steps downstream to synaptotagmin binding, and that the equilibrium constant of the binding of calcium to Munc13 controls the calcium-dependent priming process. Thus, the systematic use of the GA creates a link between specific reactions in the process of exocytosis and experimental phenotypes.

Keywords: Genetic Algorithm (GA); Exocytosis; Rate constant; Kinetic analysis; Chromaffin cell; Synaptic protein

The Src family kinase, Lyn, is activated in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors which stimulate its association with numerous other signaling molecules by Andrea Pace; Jose A. Tapia; Luis J. Garcia-Marin; Robert T. Jensen (pp. 356-365).

Src family kinases (SFK) play a central signaling role for growth factors, cytokines, G-protein-coupled receptors and other stimuli. SFKs play important roles in pancreatic acinar cell secretion, endocytosis, growth, cytoskeletal integrity and apoptosis, although little is known of the specific SFKs involved. In this study we demonstrate the SFK, Lyn, is present in rat pancreatic acini and investigate its activation/signaling. Ca²⁺-mobilizing agents, cAMP-mobilizing agents and pancreatic growth factors activated Lyn. CCK, a physiological regulator of pancreatic function, rapidly activated Lyn. The specific SFK inhibitor, PP2, decreased Lyn activation; however, the inactive analogue, PP3, had no effect. Inhibition of CCK-stimulated changes in [Ca²⁺]_i decreased Lyn activation by 55%; GFX, a PKC inhibitor by 36%; and the combination by 95%. CCK activation of Lyn required stimulation of high and low affinity CCK_A receptor states. CCK stimulated an association of Lyn with PKC-δ, Shc, p125^FAK and PYK2 as well as with their autophosphorylated forms, but not with Cbl, p85, p130^CAS or ERK 1/2. These results show Lyn is activated by diverse pancreatic stimulants. CCK's activation of Lyn is likely an important mediator of its ability to cause tyrosine phosphorylation of numerous important cellular mediators such as p125^FAK, PYK2, PKC-δ and Shc, which play central roles in CCK's effects on acinar cell function.

Keywords: Pancreas; src; src kinase; lyn; Cholecystokinin; Pancreatic secretion; Pancreatic growth; Pancreatic growth factor

Trk receptor binding and neurotrophin/fibroblast growth factor (FGF)-dependent activation of the FGF receptor substrate (FRS)-3 by Scott J. Dixon; James I.S. MacDonald; Kim N. Robinson; Christopher J. Kubu; Susan O. Meakin (pp. 366-380).

We have investigated the signaling properties of the fibroblast growth factor (FGF) receptor substrate 3 (FRS3), also known as SNT-2 or FRS2β, in neurotrophin-dependent differentiation in comparison with the related adapter FRS2 (SNT1 or FRS2α). We demonstrate that FRS3 binds all neurotrophin Trk receptor tyrosine kinases and becomes tyrosine phosphorylated in response to NGF, BDNF, NT-3 and FGF stimulation in transfected cells and/or primary cortical neurons. Second, the signaling molecules Grb2 and Shp2 bind FRS3 at consensus sites that are highly conserved among FRS family members and that Shp2, in turn, becomes tyrosine phosphorylated. While FRS3 over-expression in PC12 cells neither increases NGF-induced neuritogenesis nor activation of Map kinase/AKT, comparable to previous reports on FRS2, over-expression of a chimeric adapter containing the PH/PTB domains of the insulin receptor substrate (IRS) 2, in place of the PTB domain of FRS3 (IRS2-FRS3) supports insulin-dependent Map kinase activation and neurite outgrowth in PC12 cells. Collectively, these data demonstrate that FRS3 supports ligand-induced Map kinase activation and that the chimeric IRS2-FRS3 adapter is stimulating sufficient levels of activated MapK to support neurite outgrowth in PC12 cells.

Keywords: Abbreviations; BDNF; Brain-derived neurotrophic factor; MAPK/ERK; Mitogen-activated protein kinase/extracellular signal regulated kinase; FGF; Fibroblast growth factor; FRS2; Fibroblast receptor substrate 2; FRS3; Fibroblast receptor substrate 3; NGF; Nerve growth factor; NT-3; Neurotrophin-3; PTB; Phosphotyrosine binding; RTK; Receptor tyrosine kinase; SNT-1; Suc1-associated neurotrophic factor-1; SNT-2; Suc1-associated neurotrophic factor-2FRS3; FRS2beta; SNT-2; NGF; Trk; FGF receptor; Neurotrophin signaling

Sex-specific regulation of growth plate chondrocytes by estrogen is via multiple MAP kinase signaling pathways by J. McMillan; S. Fatehi-Sedeh; V.L. Sylvia; V. Bingham; M. Zhong; B.D. Boyan; Z. Schwartz (pp. 381-392).

Both male and female rat growth plate cartilage cells possess estrogen receptors (ERs), but 17β-estradiol (E₂) activates protein kinase C (PKC) and PKC-dependent biological responses to E₂ only in cells from female animals. PKC signaling can elicit genomic responses via mitogen activated protein kinase (MAPK) and E₂ has been shown to activate ERK MAPK in many cells, suggesting that MAPK may play a role in growth plate chondrocytes as well. We tested if E₂ increases MAPK activity and if so, whether the response is limited to female cells, if it is PKC-dependent, and if the mechanism involves traditional ER pathways. We also determined the contribution of MAPK to the biological response of growth plate chondrocytes and assessed the relative contributions of ERK, p38 and JNK MAPKs. Female rat costochondral cartilage cells were treated with E₂ and MAPK-specific activity determined in cell layer lysates. The mechanism of MAPK activation was determined by treating the cells with E₂ conjugated to bovine serum albumin (E₂-BSA) to assess if membrane receptors were involved; stereospecificity was determined using 17α-estradiol; PKC and phospholipase C (PLC) dependence was determined using specific inhibitors; and the ER agonist diethylstilbestrol, the ER antagonist ICI 182780, and tamoxifen were used to assess the role of traditional ER pathways. E₂ regulation of ERK1/2 MAPK was assessed and the relative roles of ERK1/2, p38 and JNK MAPKs determined using specific inhibitors. E₂ caused a rapid dose-dependent activation of MAPK that was greatest in cells treated for 9 min with 10⁻⁹ M hormone; activity remained elevated for 3 h. E₂'s effect on MAPK was stereospecific and comparable to that of E₂-BSA. It was insensitive to DES and ICI 182780, dependent on PKC and PLC, blocked by tamoxifen and it did not require gene transcription or translation. E₂ had no effect on ERK1 or ERK2 mRNA or protein but it caused a rapid phosphorylation of ERK1/2 at 9 min. Inhibition of ERK1/2 and p38 MAPK reduced the stimulatory effects of E₂ on alkaline phosphatase activity and [³⁵S]-sulfate incorporation. These results suggest that E₂ regulates MAPK through a sex-specific membrane-mediated mechanism that does not involve cytosolic ERs in a traditional sense and that ERK1/2 and p38 mediate the downstream biological effects of the hormone.

Keywords: 17β-Estradiol; Sex-specificity; MAP kinase; Growth plate chondrocytes; ERK; p38; JNK

Melanotransferrin stimulates t-PA-dependent activation of plasminogen in endothelial cells leading to cell detachment by Yannève Rolland; Michel Demeule; Richard Béliveau (pp. 393-401).

Tissue plasminogen activator (t-PA) is an extracellular serine protease that converts the proenzyme plasminogen into the broad-spectrum substrate serine protease, plasmin. Plasmin, one of the most potent pro-angiogenic factors, is a key element in fibrinolysis, cell migration, tissue remodeling and tumor invasion. In the present investigation, we assessed the impact of the truncated form of soluble melanotransferrin (sMTf) on plasminogen activation by t-PA and subsequent endothelial cell detachment. Co-treatment of human endothelial microvessel cells with plasminogen, t-PA and sMTf significantly increased plasmin formation and activity in the culture medium. Plasmin generated in the presence of sMTf also led to a 30% reduction in fibronectin detection within cell lysates and to a 9-fold increase within the corresponding cell medium. Moreover, the presence of sMTf increases EC detachment by 6-fold compared to cells treated only with plasminogen and t-PA. Although the addition of α₂-antiplasmin completely prevented plasmin formation and EC detachment, epigallocatechin gallate, GM6001 and a specific antibody directed against MMP-2 prevented cellular detachment without interfering with plasminogen activation. Overall, these data suggest that the anti-angiogenic properties of sMTf may result from local overstimulation of plasminogen activation by t-PA, thus leading to subsequent degradation of the Fn matrix and EC detachment.

Keywords: Melanotransferrin; Plasminogen; Tissue-type plasminogen activator; Matrix metalloproteinase; Fibronectin; Endothelial cell; Detachment

Gene expression analysis of ELF-MF exposed human monocytes indicating the involvement of the alternative activation pathway by Madeleine Lupke; Jana Frahm; Margareta Lantow; Christian Maercker; Daniel Remondini; Ferdinando Bersani; Myrtill Simkó (pp. 402-412).

This study focused on the cell activating capacity of extremely low frequency magnetic fields (ELF-MF) on human umbilical cord blood-derived monocytes. Our results confirm the previous findings of cell activating capacity of ELF-MF (1.0 mT) in human monocytes, which was detected as an increased ROS release. Furthermore, gene expression profiling (whole-genome cDNA array Human Unigene RZPD-2) was performed to achieve a comprehensive view of involved genes during the cell activation process after 45 min ELF-MF exposure. Our results indicate the alteration of 986 genes involved in metabolism, cellular physiological processes, signal transduction and immune response. Significant regulations could be analyzed for 5 genes (expression >2- or <0.5-fold): IL15RA (Interleukin 15 receptor, alpha chain), EPS15R (Epidermal growth factor receptor pathway substrate 15 — like 1), DNMT3A (Hypothetical protein MGC16121), DNMT3A (DNA (cytosine-5) methyltransferase 3 alpha), and one gene with no match to known genes, DKFZP586J1624. Real-time RT-PCR analysis of the kinetic of the expression of IL15RA, and IL10RA during 45 min ELF-MF exposure indicates the regulation of cell activation via the alternative pathway, whereas the delayed gene expression of FOS, IL2RA and the melatonin synthesizing enzyme HIOMT suggests the suppression of inflammatory processes. Accordingly, we suggest that ELF-MF activates human monocytes via the alternative pathway.

Keywords: Abbreviations; O; ₂; ⁻; super oxide anion; AML; acute monocytic leukaemia; as; antisense; C.I.; confidence interval; C3; complement component 3; CD; cluster of differentiation; CP; crossing point; DHR; dihydrorhodamine 123; E/C; ration of exposed to unexposed cells; ECM; extracellular matrix; ELF-MF; extremely low frequency magnetic fields; FITC; Fluorescein; GTP; guanosine triphosphate; HBS; HEPES-buffered saline; HIOMT; hydroxyndole-; O; -methyltransferase; HPRT; hypoxantine phosphoribosyl-transferase1; HUPO; human acidic ribosomal protein; IL; interleukin; IL10RA; interleukin 10 receptor alpha; IL15RA; interleukin 15 receptor alpha; IL2RA; interleukin 2 receptor alpha; JAK; janus kinase; LPS; lipopolysaccharide; MAPK; mitogen-activated phosphatase kinase; NADH; reduced NAD; NADPH; NAD phosphate (reduced); PDGFA; platelet-derived growth factor alpha polypeptide; PerCP; Peridinin–Chlorophyll–Protein-Complex; PMA; phorbol 12-myristate 13-acetate; ROS; reactive oxygen species; RPMI; Roswell Park Memorial Institute; RT-PCR; reverse transcriptase polymerase chain reaction; s; sense; SAM method; statistical analysis of microarray; S.D.; standard deviation; VEGF; vascular epidermal growth factorGene expression; Monocyte; Cytokine receptor; Early response gene; ELF-MF or electromagnetic field; Free radical; Alternative activation pathway

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BBA - Molecular Cell Research (v.1763, #4)