Session 12: Information Flow in Cell Oral presentations O12.1

Information flow in the nervous cell: neuroactive steroids as the nongenomic signaling molecules Ludmiła Żylińska* Department of Molecular Neurochemistry, Medical University of Łódź, Łódź, Poland *e-mail: Ludmiła Żylińska   The prevailing classical hypotheses for the mechanism of steroid hormones action postulate their genomic action. The signal is transmitted by the binding of steroids to the specific membrane, cytoplasmic and/or nuclear receptors, and next, these complexes modify the transcription of hormone-sensitive genes. Now it is proposed that the rapid steroid actions at the plasma membrane level could precede their traditional genomic mechanisms. There are two main patterns of nongenomic action. The first is linked with direct steroid interaction with the membrane proteins not being the specific receptors. The second way is the modulation of intracellular signaling systems by the steroid-receptor complex, but without contribution of transcriptional machinery. Interestingly, the hormones effects are detectable within minutes, and are insensitive to RNA and protein synthesis inhibitors. Steroids have also a strong potential to modulate processes in the CNS, entailing diverse behavioral and psychopharmacological effects in humans and other species. Steroids that are able to regulate the neuron excitability in the CNS are named “neuroactive steroids”. Apart from endocrine glands, steroid hormones can be synthesized de novo in the brain, and the term “neurosteroids” was introduced for those synthesized in the CNS. The nervous tissue appears to be under permanent genomic and nongenomic control of steroids, although their concentrations are age- and sex-dependent, and notably differ in particular areas of the brain. The nongenomic hormone action appears to be the very important alternative pathways in cellular signaling. The fast negative and positive modulatory effects were observed for g-aminobutyric acid (GABAA) receptor complex, N-methyl-d-aspartate (NMDA) receptors, G proteins-coupled receptors, and ion channels and pumps. Steroids are able to activate ubiquitous regulatory cascades such as MAPK family members, PI3 kinase, Akt/ protein kinase B, tyrosine kinases, protein kinases A and C, or phospholipases C and A2, thereby can modulate generation of various second messengers, including Ca2+, IP3, DAG, cAMP, cGMP and NO. Disturbance in nervous cell homeostasis can alter both, nongenomic and genomic mechanisms in the brain leading to relevant pathophysiological consequences. The relationship between fast steroid-induced signaling and membrane-located effectors may represent an important

area for future investigation to elucidate their potential role in neurodegenerative diseases, as well as in an eventual therapy. Acknowledgements: Supported in part by the grants No 2 P05A 03529 and 5036086-2.

Abstracts 160

O12.2

O12.3

Glomerulus and macula densa cells signaling Stefan

Angielski1*,

2007

Maciej W.

Jankowski1,2

1Laboratory

of Cellular and Molecular Nephrology, Research Centre PAS, Gdańsk, Poland, 2Department of Monitoring Therapy anf Pharmacogenetics, Medical University of Gdansk, Gdańsk, Poland *e-mail: Stefan Angielski   Macula densa cells are located within the thick ascending limb and have their basolateral membrane in contact with glomerular mesangial cells, which, in turn, are in a direct contact with smooth muscle cells of afferent arteriole. Macula densa and mesangial cells are involved in an intrinsing renal mechanism that stabilizes glomerular filtration rate (GFR) at single nephron level, termed tubuloglomerular feedback (TGF). Macula densa cells are able to sense changes in luminal NaCl concentration and to transmit signal that ultimately constricts the afferent arteriole and reduces glomerular filtration rate. It has been shown that increases in luminal [NaCl] induces macula densa cells to release increasing amounts of ATP across the basolateral membrane. Released ATP to extracellular space affects glomerular cells (mesangial and podocytes) and smooth muscle cells which expressed P2 receptors. Glomerular cells expressed also an active ecto-nucleotidases which are able to degrade of ATP and finally to terminate ATP signaling. ATP degradation leads to the formation of adenosine and activation of P1 receptors, which can also play a role in TGF signaling. The release of ATP via channels and the presence of ecto-nucleotidases provide suggestion for a rapid local signaling process with fast on/off kinetics. It should be emphasized that macula densa cells are very rich in mitochondria and express unique low activity of basolateral Na,K-ATPase. It may suggest that high capacity to generate ATP is due to a high turnover of ATP involved in local signaling process. From the other hand metabolic signal from macula cells may affects the tension of glomerular cells. The tension of mesangial cells and podocytes may influence GFR by affecting the glomerular ultrafiltration coefficient, that is, contraction/relaxation of mesangial cells and/or podocytes may lead to decrease/increase in GFR. On the basis of the available data and their interpretation, we have suggested the GFR regulation does not occur only at the afferent arteriole level but may involve the intraglomerular elements that can modify glomerular capillary filtration and its volume.

Information flow in the muscle cell: the effect of calcium signal on carbohydrate metabolism regulation in skeletal muscle cell Andrzej Dzugaj* Faculty of Biological Sciences, Wrocław University, Wrocław, Poland *e-mail: Andrzej Dzugaj   In skeletal muscle, calcium not only triggers muscle contraction but also exerts pleiotropic effects on carbohydrate metabolism. Calcium activates glycogen phosphorylase-accelerating glycogenolysis, induces GLUT 4 and activates pyruvate dehydrogenase, thus accelerating glycolysis. Calcium also inhibits glyconeogenesis. It has been reported that up to 50% of lactate generated in muscle tissue is converted in situ to glycogen. This process was called glyconeogenesis to distinguish it from gluconeogenesis, which takes place in liver and results in glucose release. Regulatory enzyme of gluconeogenesis and glyconeogenesis is fructose-1,6-bisphosphatase (FBPase) catalyzing hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and Pi. Liver and muscle isozyme have been found in vertebrate tissues. Kinetic properties of both isozymes are virtually the same, both require divalent metal ions like magnesium to their activity and both are activated by monovalent cations and inhibited by fructose-2,6-bisphosphate. Recently we have found that calcium is the strong inhibitor of the muscle FBPase which binds the calcium ions in a cooperative manner. The determined I0.5 of the muscle isozyme toward calcium was 0.6 μM. On the contrary, the liver isozyme is practically insensitive to this ions. Investigating the muscle FBPase we found that the enzyme interacts with the muscle aldolase and α-actinin. We found that, in vivo FBPase colocalizes with aldolase and α-actinin on the Zline in myocytes making a glyconeogenic metabolon. Increase of calcium concentration results in dissociation of FBPase from the Z-line, disintegration of FBPase-aldolase complex and in effect inhibition of the enzyme. At rest decrease of calcium concentration enables association of FBPase with aldolase and colocalization of both enzymes with α-actinin on the Z-line so, glyconeogenesis can proceed. Searching for the origin of the high sensitivity of the muscle FBPase toward calcium we found that in the liver isozyme, Gln69 is present which is substituted with Glu69 in the muscle isozyme. Hypothetically the single point mutation Gln→Glu resulted in dramatic change of the former isozyme kinetic property making the new isozyme highly sensitive to calcium ions thus enabling the regulation of glyconeogenesis by calcium signal.

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O12.4

O12.5

Flow of information in cancer cell based on melanoma and prostate cancer

Signal transduction pathways responsible for cytoskeletal activity regulation in protozoan cells

Piotr M. Laidler*, Dorota Ciołczyk, Joanna D. Dulińska, Dorota Gil, Jędrzej Małecki

Paweł Pomorski*, Hanna Fabczak, Wanda Kłopocka, Stanisław Fabczak

Department of Medical Biochemistry, Collegium Medicum, Jagiellonian University, Kraków, Poland *e-mail: Piotr M. Laidler  

Nencki Institute of Experimental Biology, Warszawa, Poland *e-mail: Paweł Pomorski  

Akt kinase is known to play a central role in many cellular processes including proliferation, differentiation, migration (adhesion) and apoptosis. Survival signals induced by several receptors are mediated mainly by phosphatidylinositol-3-kinase (PI3K)/Akt kinase. Constitutive activation of Akt kinase is frequently described in many types of human cancers. Relatively less recognized are mechanisms that involve Akt kinase in signal transduction mediated through adhesion molecules including cellcell and cell-extra cellular matrix proteins interactions. We were able to show that two adhesion molecules – α3β1 integrin and N-cadherin are likely involved in signal transduction that recruits Akt kinase. Laminin-5 (LN-5)integrin dependent signaling pathway activates matrix metalloproteinase (MMPs) in human melanoma cells. Increased level of phospho-AKT (pAkt, Ser-473) in the cells cultured on LN-5 was observed. This signaling pathway, necessary for migration of melanoma cells, was shown to involve PI3-K/Akt activity as the addition of the PI3-K specific inhibitor, LY294002, decreased the level of pAkt (Ser-473) and in parallel the expression and activity of MMP-2 and 9 in melanoma cells. Independent study on the role of N-cadherin — which upon cancer progression replaces E-cadherin in number of cells — showed that silencing of its expression by siRNA in melanoma cells reduced phosphorylation of Akt (Ser473) and in parallel significantly decreased cell proliferation (50–70%). N-cadherin is known to promote in vitro migration and is likely involved in inhibition of apoptosis in melanoma cells. We have proven that blocking of the PI3K/Akt pathway with LY294002 and PI3K/AktN-cadherin-β-catenin-actin interactions with cytochalasine D significantly increases caspase-3 activity in human melanoma cells. We recently postulated that beta-catenin plays important role in sustaining survival signals in prostate cancer cells. The increased expression of E-cadherin and/or decreased expression of N-cadherin was accompanied by reduced expression of beta-catenin and decreased proliferation of prostate cancer cells and the effect likely depended on their androgen sensitivity [1]. Our most recent results indicate that Akt promotes androgen-independent survival of prostate cancer cells by modulating the expression and activation of the androgen receptor (AR). This pathway seems to involve beta-catenin, too. Therefore, we try to look for the possible interaction (cross-talk) between Akt kinase and beta-catenin signaling pathways which might be crucial for development and progression of cancer. Reference: 1. Laidler P, Dulińska J, Mrozicki S (2007) Arch Biochem Biophys in press.

Cytoskeleton as well as molecular-motor proteins existing in protista cells are responsible for both: generating their movement and maintaining the cell shape. The main players here are cytoskeletal proteins: tubulin and actin, accompanied by molecular motors: dyneins, kinesins and myosins. This system works under the control of numerous regulatory proteins and smaller molecules such as calcium ions and cyclic nucleotides. The motile system in ciliate protists is based on axoneme, which is the most representative and conservative structure consisting of microtubule and dynein. Extracellular stimuli are converted in these eukaryotic cells through signal transduction mechanisms to generate intracellular second messengers, Ca2+ and cyclic nucleotides. These molecular signals, amongst other, control the ciliary locomotor system by modulating the activity of axoneme, changing the direction and frequency of effective ciliary beating. The primary role in regulating the mechanisms of axoneme motility by second messengers is accomplished by phosphorylation and dephosphorylation of axoneme proteins. Ca2+ may also influence the levels of cAMP and cGMP by controlling the activity of cAMP and cGMP cyclases. In addition, in regulation of axoneme activity voltage-dependent ion channels (K+ and Ca2+) are involved, thus affecting the cell membrane potential in these cells. Quite another mean of movement is used by crawling amoebae, Amoeba proteus, Acanthamoeba castellanii or ameboic fragments of Dictyostelium discoideum. The main players of motility regulation in those cells are calcium ions and RhoA family small G proteins. RhoA regulates activity of effector proteins, including Rho dependent kinase (ROCK). Those effector proteins regulate activity of actomyosin. As in case of tissue cells, actomyosin contraction is regulated in protista in calcium dependent manner, and rise of cytoplasmic calcium leads to the actomyosin contraction. Contrary to tissue cells the different is way of myosin II phosphorylation in regulation of this process. In multicellular organisms, phosphorylation of MLC leads to its sensitization for calcium signal. In A. castellanii, D. discoideum and probably A. proteus it is myosin heavy chain phosphorylation what desensitizies actomyosin for calcium signal. Nevertheless the rest of signaling machinery works in the same way, leading to the reverse answer to known signaling pathways, and situation where inhibition of RhoA/ROCK signaling leads to activation of actomyosin contractility.

Abstracts 162

O12.6 Signal transduction in yeast upon stress conditions Iwona Wojda1*, Jan-Paul Bebelman2, Teresa Jakubowicz1, Marco Siderius2 1Department

of Invertebrate Immunology, Maria CurieSklodowska University, Lublin, Poland, 2Department of Biochemistry and Molecular Biology, Vrije Universiteit Amsterdam, Amsterdam, Netherlands *e-mail: Iwona Wojda   The natural environment of unicellular organisms like yeast does not provide constant growth conditions. Yeast cells are challenged by changes in e.g., temperature, osmolarity, pH, the presence of reactive oxygen intermediates, the availability of nutrients or a combination of these agents. In order to continue growth, cells have to sense and properly respond to these changes to adapt their growth potential. Adverse growth conditions induce the general stress response and specific responses. The former evoke common molecular responses such as expression of heat shock proteins (Hsp12, Hsp26, Hsp104), degradation of damaged proteins as a result of increased expression of polyubiquitin gene (UBI4), increase in trehalose synthesis (higher expression of TPS2 encoding trehalose phosphate phosphatase) etc. The general stress response is sometimes called methabolic stress and is regulated by Ras-cAMP signalling pathway. Apart from inducing general stress response differential extracellular conditions trigger also specific pathways regulating expression of genes necessary under certain circumstances. Sacharomyces cerevisiae posses at least five MAP kinase pathways (Mitogen Activating Protein) responding to different extra-or intracellular stimuli. Among them are: cell integrity (protein kinase C) pathway induced by elevated temperature, morphogenetic events, hypo-osmotic shock or cell wall perturbation, HOG (High Osmolarity Glycerol) pathway activated upon hyper-osmotic conditions, Filamentous/Invasive Growth - FG pathway and Sporulation pathway, activated upon starvation and Mating pathway, activated by mating pheromone. Our focus is on the interplay between signalling pathways activated by the osmotic stress and elevated temperature. We show that the osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature and the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for the ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37oC, suggesting the additional role of Ste11p in the maintenance of cellular integrity. This phenotype can be suppressed by genes involved in cell wall maintenance and genes increasing internal glycerol level. Also we show that sensing and response to osmotic stress is temperature-dependent. For example activation Hog1p MAP kinase, which occurs under hyperosmotic condtions, is very transient at 37oC in comparson to optimal growth temperature (28oC), and activation of Mpk1p/ Slt2p — the MAP kinase activated at elevated temperature is inhibited under hyperosmotic conditions and this phenomenon is independent of HOG-pathway activity.

2007

Additionally, accumulation of intracellular glycerol, the osmolyte in yeast, is different under the same osmotic stress at different temperatures. Finally we discuss the role of intracellular glycerol in the maintenance of cellular integrity in Saccharomyces cerevisiae.

42nd Meeting of the Polish Biochemical Society Vol. 54

O12.7

Posters

Protein kinases involved in abiotic stress response in plants

P12.1

Grażyna

Muszyńska*,

Grażyna Dobrowolska

Institute of Biochemistry and Biophysics PAS, Warszawa, Poland *e-mail: Grażyna Muszyńska   Several families of protein kinaases are involved in plants stress signaling. Among them are kinases common for all eukaryotes (e.g. kinases consisting MAPK cascades), as well as kinases specific for plants. In our laboratory we have identified, cloned and partly characterized two specific for plants protein kinases (ZmCPK11 and NtOSAK) which are involved in abiotic sress signal transduction. In maize leaves the activity of the expression of calcium dependent protein kinase classified as ZmCPK11 is activated by wounding (Szczegielniak et al., 2005). Activity and the level of ZmCPK11 transcript is also increased in non-injured neighboring leaves, what suggests that the maize protein kinase in involved in a systemic response to wounding. The recent evidences demonstrate that ZmCPK11 is the integral component of wound induced jasmonic acid dependent stress response. In tobacco cells (BY-2) was identified a protein kinasse activated rapidly in response to osmotic stress (Mikołajczyk et al., 2000). The kinase, named as NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), belongs to SNF1-related kinase 2 (SnRK2) subfamily. Phosphorylation is involved in activation NtOSAK. We mapped the regulatory phosphorylation sites of NtOSAK by mass spectrometry (Burza et al., 2006). In BY-2 cells subjected to osmotic stress Ser-154 and Ser-158 is phosphorylated. It correlates with NtOSAK activity, what indicates that NtOSAK is regulated by reversible phosphorylation of these residues in vivo. References: Burza AM, Pękala I, Sikora J, Siedlecki P, Małagocki P, Bucholc M, Koper L, Zielenkiewicz P, Dadlez M, Dobrowolska G (2006) Nicotiana tabacum osmitic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop. J Biol Chem 281: 34299–34311. Mikołajczyk M, Awotunde OS., Muszyńska G, Klessig DF, Dobrowolska G (2000) Osmotic stress induces rapid activation of a salicylic acid-induced protein kinase and a homolog of protein kinase ASK1 in tobacco cells. Plant Cell 12: 165–178. Szczegielniak J, Klimecka M, Liwosz A, Ciesielski A, Kaczanowski S, Dobrowolska G, Harmon AC, Muszyńska G (2005) A wound responsie and phospholipid-regulated maize calciumdependent protein kinase. Plant Physiol 139: 1970–1983.

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The activity of p38 MAPK, ERK1/2, JNK in PC12 cells with altered Ca2+ homeostasis Tomasz Boczek*, Anna Kozaczuk, Joanna Taha, Christos Kargas, Ludmiła Żylińska Department of Molecular Neurochemistry, Medical University of Łódź, Łódź, Poland *e-mail: Tomasz Boczek   Pituitary adenylate cyclase-activating polypeptide (PAAP) and nerve growth factor (NGF) have been shown to promote neurite outgrowth and inhibit proliferation of rat pheochroocytoma (PC12) cells. PACAP- and NGFmediated neuritogenesis is related to the activation of variety of downstream processes involving stimulation of specific kinases and kinases-dependent pathways. PACAP and NGF regulate common and distinct mechanisms to control PC12 cell differentiation suggesting their complementary role. The activity of plasma membrane calcium pump (PMCA) controls the intracellular Ca2+ and its role is prominent in neurite outgrowth. PMCA exists in 4 isoforms, with various distribution among tissues from neuron-specific PMCA2 and 3 to PMCA1 and 4 broadly distributed in nearly all tissues. Our previous research showed that neuronal differentiation of PC12 may be induced by signals other than extracellular. The neuronspecific isoforms mRNA’s antisense treatment affected the basal level of Ca2+, generating the metamorphosis of PC12 into pseudo-neuronal phenotype. The level of Ca2+ in cell lines carrying desired antisense was increased, as detected previously. The aim of this study was to examine the activity changes of p38 MAPK, ERK1/2 and JNK under the condition of altered calcium homeostasis generated by specific blocking of PMCA2, PMCA3 or both isoforms expression. We observed differences in the activity of examined kinases as well as alteration in kinasesdependent pathways. This may suggest that increase in Ca2+ level can serve as a switcher of kinases activity in a different manner than under calcium homeostasis. In the absence of upstream signals the regulation of kinases activity may also be altered, however this requires further study. Acknowledgements: Supported by the grants No: PBZ-MIN-012/P04/2004 and 2/ P05A/03529 from the Ministry of Education and Science, and 503-6086-02 from the Medical University of Lodz.

Abstracts 164

P12.2 Phosducin-like protein and chaperonin CCT are associated with Tetrahymena tubulin involved in cilia biogenesis Cezary Bregier*, Katarzyna Sobierajska, Bożena Groszyńska, Hanna Fabczak, Stanisław Fabczak Nencki Institute of Experimental Biology PAS, Warszawa, Poland *e-mail: Cezary Bregier   Microtubules are indispensable dynamic structures that contribute to many essential biological functions including cell division, intracellular transport and maintenance of cell shape or cell motility. Assembly of the native αβtubulin heterodimer, the subunit that polymerizes to form microtubules, requires participation of chaperonin CCT. The cytosolic chaperonin CCT is a heterooligomeric complex of about 900 kDa present in all eukaryotic cells. Increasing number of proteins has been shown to interact with CCT. One of them is phosducin-like protein (PhLP) which may play regulatory function in this complex. However, the potential role of PhLP in tubulin folding mechanism has not been explicated so far. It has been shown in Tetrahymena thermophila ciliates that CCT protein is associated with microtubule structures and is involved in cilia biogenesis. These cells covered with thousands of cilia can be relatively easy deciliated therefore they present excellent model for study putative function of PhLP as a regulator of CCT-mediated tubulin folding during cilia regeneration. For the above reasons the purpose of this study was to verify if PhLP present in these ciliates may be engaged in the control of the tubulin folding. To achieve this immunological methods, cell fractionation and protein expression were employed. For investigation of PhLP and CCT expression during cilia regeneration, samples of total protein extract were analyzed by Western blot using antibodies specifically recognizing PhLP and CCTε. These experiments showed that levels of PhLP and CCTε decreased significantly after deciliation and then expression of these proteins started to increase reaching after 60 min the higher levels than in control. Cilia after this period were almost fully regenerated. Localizations of CCTε and PhLP in control, deciliated and reciliating cells were accomplished by laser confocal microscopy and cell fractionation method. To attain this tested ciliates were fractionated in 5 parts: cytosol (Sm), microsomal (Pm), cilia (Cc), soluble (Str) and insoluble (Ptr) in TritonX-100 cortex fractions. The experiments evidenced that major amount of PhLP in control cells was localized mainly in Pm and Str fractions. After cilia removing PhLP was detected mostly in Str fraction and expression of this protein increased with time. CCTε in control cells was detected in Cc, Str and Pm cell fractions and after deciliation partial translocation of the CCTε to Sm fraction was observed. Immunocytochemical experiments evidenced that PhLP and CCTε were colocalized with β-tubulin in control and deciliated cells. Observed changes in localization of PhLP and CCTε and colocalization of these proteins with β-tubulin in control and deciliated cells can indicate that in Tetrahymena thermophila cili-

2007

ate both proteins may be involved in process of tubulin folding and/or microtubule assembly.

Acknowledgements: This study was supported by grants 2P 04C 014 27 from the Ministry of Science and Higher Education.

42nd Meeting of the Polish Biochemical Society Vol. 54

P12.3

P12.4

Role of androgen receptor in prostate cancer cells progression

Constitutive signalling of GPR40 & GPR120 receptors

Joanna D. Dulińska*, Piotr M. Laidler

Adam I. Cygankiewicz1,2*, Birgitte Holst2, Thue W. Schwartz2,3

Department of Medical Biochemistry, Collegium Medicum, Jagiellonian University, Kraków, Poland *e-mail: Joanna D. Dulińska   The androgen-signaling pathway plays a critical role in the regulation of prostate cancer cell growth and survival. Consequently, androgen ablation has been used as an effective treatment for the majority of advanced prostate cancers. Androgen receptor (AR), like other members of the steroid receptor superfamily, functions as a ligand-activated transcription factor, controlling the expression of genes involved in cell proliferation, migration, differentiation and death. Increasing cellular level of AR not only intensifies androgen-induced cell proliferation but also increases the sensitivity of prostate cancer cells to androgens, allowing tumor cells to grow and migrate in a low androgen environment. To understand whether and how the AR is critical in prostate carcinogenesis, we used siRNA constructs to observe the AR’s effect on androgen-induced transcription, cell proliferation and migration. Three AR siRNA constructs that contained 21-mer sequences derived from different coding regions of the human AR (AMBION) all showed specific silencing of AR expression. Treatment of LNCaP cells with siRNA for AR significantly reduced the expression of prostatic cancer markers as prostate specific antigen and prostatic acid phospatase. RT-PCR, immunoprecipitation and Western blot analysis were used to observe possible interaction of the androgen receptor with β-catenin in prostate cancer cells and AR’s function in β-catenin pathway. The high expression of β-catenin and over expression of prostatic markers was significantly associated with progression of carcinogenesis and demonstrated a dose- and time-dependence on steroid hormones (testosterone, DHT, estradiol). Free unphosphorylated β-catenin was translocated into the nucleus what was accompanied by increased cell migration as well as proliferation in androgen dependent cancer cell line (Boyden chamber; Cell proliferation, ELISA, BrdU; Violet crystal). Simultaneously phospho-β-catenin (ser33/37/Thr41 and thr41/Ser45) blocking peptides (Cell Signaling) were applied to confirm unphosphorylated and phosphorylated level of β-catenin after treatment the cells with androgen after the use siRNA for AR. Based on the studies on AR-expressing cells (LNCaP) and non-AR-expressing cells (PC3, Du145) we postulate that the AR can have an effect on passage of the β-catenin into the nucleus when exposed to exogenous androgen. These observation suggest the role of β-catenin in the regulation of AR function and its role in prostate cancer progression.

165

1Department

of Cytobiochemistry, University of Łódź, Łódź, Poland, 2Laboratory for Molecular Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark, 37TM Pharma A/S, Hørsholm, Denmark *e-mail: Adam I. Cygankiewicz   GPR 40 and GPR 120 are members of 7 trans-membrane receptor family. 7TM receptors are ubiquitously expressed in human tissues. GPR40/120 among others, are expressed in the gastrointestinal tract and in the endocrine pancreas and are believed to be involved in the control of the secretion of for example insulin and glucagon like peptide-1. Growing numbers of these receptors are identified as “constitutively active” – they signal through natural signaling pathways in absence of ligand. Interfering or modulation of this phenomenon proves to be interesting as new drug target. To understand mechanisms of GPR40 & GPR 120 intracellular signalization we have conducted studies which allowed identification of G protein subunits responsible for trafficking of GPR40/120 signals. We have studied ligand independent (constitutive) signaling through CREB and SRE pathways in cells after exposition to forskolin and pertussis toxin. This procedure employs co-transfection of cells with promiscous G proteins which enhances cellular response after receiving signals from receptors. Proper recognition of proteins involved in signal transduction is necessary for development of sensitive and accurate screening of compounds which are suggested ligands for studied receptors. Recently it has been proposed that GPR40 constitutive activity was an effect of endogenous ligands (e.g. fatty acids) being bound to BSA. We have conducted a series of experiments in which this possibility was checked. Obtained results indicated that fatty acids bound to BSA do not influence activity of GPR 40 receptors. More in-depth knowledge about pharmacological features of these receptors may contribute to development of more efficient treatment of hyperglycemias and other diseases like obesity, hypertension and cancer in which GPR 40 & GPR 120 seem to be involved.

Abstracts 166

P12.5

P12.6

The role of Toll-like receptor 2 on murine macrophages in signaling regulating maturation of phagosome containing dead Mycobacterium avium 101

Fast effect of estradiol on calcium transport in PC12 cells

Adam Iwanicki1,2*, Jeffrey Schorey2 1Medical

University of Gdańsk, Gdańsk, Poland, 2University of Notre Dame, Notre Dame, United States *e-mail: Adam Iwanicki   A hallmark of pathogenic mycobacteria is their ability to block phagosome maturation. The vacuole that pathogenic mycobacteria reside within can be characterized by limited proteolytic activity, lowered acidity and lack of specific maturation markers such as Rab7, syntaxin-6 and LAMP-1. Although we are beginning to appreciate how pathogenic mycobacteria block phagosome maturation, there still remain gaps in our understanding. We have been conducting studies to address TLR2 role in regulating mycobacterial phagosome maturation based on published data indicating that TLR2 and -4 promote trafficking of phagocytosed bacteria to a phagolysosomal compartment. We did not observe any differences between murine bone marrowderived macrophages isolated from TLR2–/– or wild type mice in the phagocytosis or trafficking of live Mycobacterium avium. However, phagosomes containing heat-killed M. avium showed delayed acidification in TLR2–/– compared to wild type macrophages and a lack of lysosomal associated membrane protein-1 staining in the knockout macrophages. The belated phagosome acidification seen in TLR2–/– macrophages may be due to an observed delay in the proton ATPase recruitment. Pathogenic Mycobacteria are known to block mobilization of calcium from intracellular compartments leading to inhibition of Ca2+ signaling, vital for the process of phagosome maturation. Moreover, it has recently been shown that TLR2 can activate these signaling pathways. We have analyzed the role of TLR2 receptor in calcium-dependent signaling pathway in context of phagosome maturation. We are continuing to characterize the signaling pathways that may originate from TLR2 receptor and regulate maturation of phagosomes containing dead M. avium 101. Obtained results should help in understanding the role of this receptor in controlling of mycobacterial infections.

2007

Anna Kozaczuk*, Joanna Taha, Tomasz Boczek, Christos Kargas, Ludmiła Żylińska Department of Molecular Neurochemistry, Medical University of Łódź, Łódź, Poland *e-mail: Anna Kozaczuk   Intracellular Ca2+ signaling is a key regulator of proliferation, cell cycle progression and apoptosis. The plasma membrane Ca2+-ATPase (PMCA), encoded by four separate genes, is a crucial controller of intracellular calcium signaling. Steroid hormones belong to physiologically important direct or indirect regulators of calcium pump activity. Steroids produced in the CNS (neurosteroids) in most cases are neuroactive and play an important role in many brain functions through genomic and non-genomic action. While action of steroids at the genome requires a time period from minutes to hours limited by the rate of protein biosynthesis, the modulatory effects of neuroactive steroids are faster occurring events within milliseconds to seconds. Thus, the genomic and nongenomic effects of steroids within the central nervous system provide the molecular basis for a broad spectrum of steroid action on neuronal function and plasticity. Neurosteroids appears to participate in the membrane enzymes activity regulation, playing crucial role in maintenance of neuronal calcium homeostasis. PC12 cell line is a suitable and frequently used model for the study on neurotransmission, including Ca2+ signaling. To evaluate the potential role of neuron-specific PMCA isoforms in maintenance of calcium homeostasis, we have constructed the stably transfected PC12 cell lines, with suppressed expression of PMCA 2 and PMCA3. The aim of presented study was to elucidate the influence of selected steroid hormones on PMCA transport activity in control and transfected PC12 cell lines. Analysis of short term action of 17-α-estradiol and 17-β-estradiol at physiological and pharmacological concentrations showed that transport of calcium ions is modulated in non genomic manner on plasma membrane level, and this effect appeared to be dependent on PMCAs composition. Acknowledgements: Supported by the grants No: PBZ-MIN-012/P04/2004 and 2/ P05A/03529 from the Ministry of Education and Science, and 503-6086-2 from the Medical University of Lodz.

42nd Meeting of the Polish Biochemical Society Vol. 54

P12.7 Proteins induced by nicotine in Caenorhabditis elegans Robert Sobkowiak*, Andrzej Lesicki Department of Cell Biology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland *e-mail: Robert Sobkowiak   Nicotine, the primary addictive substance in tobacco, induces profound behavioral responses in mammals, but the underlying genetic mechanisms are not well understood. Caenorhabditis elegans exhibits behavioral responses to nicotine that parallel those observed in mammals, including acute response, tolerance, withdrawal, and sensitization. The worms have a simple, well-characterized nervous system, so they are well suited for investigating how specific neurotransmitters, receptors, and signaling molecules function within the context of the nervous system to produce behavior. The aim of this study was to detect the proteins related to nicotine addiction in Caenorhabditis elegans. These proteins may be useful in revealing the molecular mechanism of nicotine addiction and in isolating the potential drug targets for nicotine addiction-associated disease therapy. The experiments were carried out on C. elegans wild-type strain Bristol (N2). The worms were grown on S medium by using concentrated E. coli OP50 as a food source. The liquid cultures of C. elegans were treated with nicotine (30 mM) for 1 h. The animals were homogenized in 50 mM Hepes-KOH (pH 7.6), 1 mM EDTA, 140 mM KCl, 0.5% NP-40, and 10% glycerol. The accumulation of proteins was analyzed by 16-BAC/SDS/PAGE for resolution of integral membrane proteins (Hartinger et al. (1996) Analyt Biochem 240: 126–133). The first-dimension separation occurs according to molecular weight in an acidic discontinuous PAGE system (pH 4.1–2.1) using cationic benzyldimethyl-n-hexadecylammonium chloride (16-BAC) as a detergent. Because proteins show slightly different migration properties, as compared to the SDS/PAGE system, the resolution can be improved by subsequent second-dimension SDS/PAGE. Nicotine induced changes in protein pattern. The most pronounced effect of acute exposure of naive animals to nicotine was the preferential accumulation of polypeptides with a molecular weight of 36 kDa. Further research has been undertaken to identify proteins sequences in selected spots by mass spectrometry.

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