BBA - Molecular Cell Research (v.1538, #2-3)
Structural and functional aspects of filamins by Arjan van der Flier; Arnoud Sonnenberg (99-117).
Filamins are a family of high molecular mass cytoskeletal proteins that organize filamentous actin in networks and stress fibers. Over the past few years it has become clear that filamins anchor various transmembrane proteins to the actin cytoskeleton and provide a scaffold for a wide range of cytoplasmic signaling proteins. The recent cloning of three human filamins and studies on filamin orthologues from chicken and Drosophila revealed unexpected complexity of the filamin family, the biological implications of which have just started to be addressed. Expression of dysfunctional filamin-A leads to the genetic disorder of ventricular heterotopia and gives reason to expect that abnormalities in the other isogenes may also be connected with human disease. In this review aspects of filamin structure, its splice variants, binding partners and biological function will be discussed.
Keywords: Filamin; Gelation factor; Actin cross-linking; Signal transduction;
Involvement of calcium signaling in the fibronectin-stimulated macrophage recognition of oxidatively damaged erythrocytes by Masatoshi Beppu; Masayoshi Azuma; Noriaki Maruyama; Kiyomi Kikugawa (119-128).
Macrophages recognize oxidatively damaged autologous erythrocytes, and cell surface fibronectin of macrophages enhances the recognition (Beppu et al., FEBS Lett. 295 (1991) 135–140). In the present study, mechanisms of enhanced macrophage recognition of oxidatively damaged erythrocytes by fibronectin were investigated. Monolayers of thioglycollate-induced mouse peritoneal macrophages with cell surface fibronectin recognized autologous erythrocytes oxidized with an iron catalyst ADP/Fe3+. The macrophage recognition of the oxidized erythrocytes was inhibited partially by pretreatment of the macrophage monolayers with a Ca2+ channel blocker (diltiazem), calmodulin inhibitors (W-7, trifluoperazine, chlorpromazine and dibucaine), an inhibitor of myosin light chain kinase (ML-9), a microfilament formation inhibitor (cytochalasin B), phospholipase A2 inhibitors (4-bromophenacyl bromide, mepacrine) and cyclooxygenase inhibitors (indomethacin and aspirin). Monolayers of macrophages depleted of fibronectin by trypsinization lost the ability of recognizing oxidized erythrocytes, but acquired the ability when stimulated with a fibronectin-coated coverslip. The recognition of fibronectin-stimulated trypsinized macrophages was also inhibited by the above inhibitors. On treatment with Ca ionophore A23187, trypsinized macrophages acquired the ability to recognize oxidized erythrocytes. The recognition of Ca ionophore-stimulated trypsinized macrophages was inhibited by the above inhibitors except the Ca2+ channel blocker. These results indicate that the Ca2+ signaling including Ca2+ influx, calmodulin activation and myosin light chain phosphorylation are involved in the fibronectin stimulation of the recognition of macrophages for oxidized erythrocytes. Involvement of microfilament formation and arachidonate cascade in the fibronectin stimulation was also suggested.
Keywords: Fibronectin; Macrophage; Oxidized erythrocyte; Calcium signaling; Arachidonate cascade;
Wnts differentially regulate colony growth and differentiation of chondrogenic rat calvaria cells by Clemens Bergwitz; Thomas Wendlandt; Andreas Kispert; Georg Brabant (129-140).
The wingless- and int-related proteins (Wnts) have an important role during embryonic development and limb patterning. To investigate their function during chondrocyte differentiation, we used NIH3T3 cells producing seven members of the Wnt family and secreted frizzled-related protein (sFRP-2) for co-culture experiments with the rat chondrogenic cell line pColl(II)-EGFP-5. Pilot experiments showed a negative effect of Wnt-7a on the proliferation of three rodent chondrogenic cell lines, RCJ3.1(C5.18), CFK-2, and C1. To establish a reporter system for chondrogenic differentiation we then produced a stably transfected chondrogenic cell line based on RCJ3.1(C5.18) for further experiments, which expresses green fluorescence protein (EGFP) under the collagen type II promoter (pColl(II)-EGFP-5). This cell line permits convenient observation of green fluorescence as a marker for differentiation in life cultures. The colony size of this cell line in agarose suspension cultures was reduced to 20–40% of control, when exposed to Wnt-1, 3a, 4, 7a, and 7b for 14 days. Similarly, reporter gene expression and the synthesis of cartilage-specific proteoglycans were inhibited by this group of Wnts. In contrast, pColl(II)-EGFP-5 cells exposed to Wnt-5a and Wnt-11 reached 140% of control, and reporter gene expression and proteoglycan synthesis were stimulated. The effects of Wnt-7a and Wnt-5a were additive in pColl(II)-EGFP-5 cells and some but not all Wnt effects were antagonized by the inhibition of proteoglycan sulfation with chlorate, by sFRP-2, which may modulate Wnt receptor binding, or by inhibitors of protein kinase C. These results suggest two functional Wnt subclasses that differentially regulate proliferation and chondrogenic differentiation in vitro which may have implications for cartilage differentiation in vivo. Since some, but not all Wnt effects were sensitive to inhibitors of proteoglycan synthesis or protein kinase C, multiple modes of signal transduction may be involved.
Keywords: Collagen type II gene expression; Wnt family (Wnt-1, Wnt-3a, Wnt-4, Wnt-7a, Wnt-7b, Wnt-5a, Wnt-11); RCJ3.1(C5.18) rat calvaria cell; Chlorate; Secreted frizzled-related protein; Differentiation; Chondrocyte; Protein kinase C;
Mannose 6-phosphate-independent endocytosis of β-glucuronidase by human fibroblasts by Alfonso González-Noriega; Colette Michalak; Jose Raymundo Cruz-Perez; Felipe Masso (141-151).
Prior work has shown that endocytosis of bovine β-glucuronidase by human fibroblasts can be mediated by the existence of a Man6P-independent receptor for the recapture and targeting to lysosomes. In this study, we have isolated a peptide (IIIb2) from pronase digested bovine β-glucuronidase that behaved as competitive inhibitor of the endocytosis of bovine β-glucuronidase by human fibroblasts. This peptide contained a Ser–X–Ser sequence, where X is probably a posttranslational modified Trp. Antibodies raised against this peptide impaired the endocytosis of the bovine but not the human β-glucuronidase, implying that the new recognition marker for the endocytosis of acid hydrolases might reside in a single discrete stretch of amino acid sequence. On the other hand, bovine β-glucuronidase has been shown to bind specifically to receptors of human fibroblast membranes. The binding was saturable, divalent cation-dependent and was competitively inhibited by the IIIb2 peptide, but not by mannose 6-phosphate. Results presented suggested an interplay between manganese concentrations, temperature and pH on the dissociation of the β-glucuronidase-receptor complexes. All together, these data reinforce the presence of two endocytic systems for the recapture and targeting of β-glucuronidase in human fibroblasts.
Keywords: Mannose 6-phosphate; Cation-independent M6P receptor; β-Glucuronidase; Acid hydrolase targeting; Acid hydrolase endocytosis;
Mannose 6-phosphate-independent endocytosis of β-glucuronidase by Alfonso González-Noriega; Colette Michalak (152-161).
A new binding protein, which recognizes a specific peptide sequence from pronase digested bovine β-glucuronidase, has been isolated from bovine liver membranes. Prior work has shown that this peptide (IIIb2) contains a Ser–X–Ser sequence, where X might be a posttranslational modified Trp. This receptor was detergent-extracted from total bovine liver membranes and purified by affinity chromatography on a bovine β-glucuronidase–Sepharose and a IIIb2 peptide–Sepharose column. Binding of bovine β-glucuronidase to the isolated receptor requires divalent cations, and their presence was necessary to maintain the receptor–ligand complex. Only the peptide sequence containing the fraction IIIb2 was able to impair the binding of the bovine enzyme to the receptor, no other peptide from bovine β-glucuronidase had an effect on binding. When analyzed by SDS–PAGE under reducing conditions, two bands were observed, a major band of 78 kDa and a faint band of 72 kDa. Rabbit antibodies against this binding protein revealed the presence of the 78 kDa protein in membranes from bovine liver, human and bovine fibroblasts. These antibodies impaired human fibroblasts endocytosis of the bovine but not of the human β-glucuronidase, which is taken up by a 300 kDa receptor that recognizes phosphomannosyl moieties in the enzyme.
Keywords: Mannose 6-phosphate; Cation-independent M6P receptor; β-Glucuronidase; Acid hydrolase targeting; Acid hydrolase endocytosis;
Molecular properties of apelin: tissue distribution and receptor binding by Yuji Kawamata; Yugo Habata; Shoji Fukusumi; Masaki Hosoya; Ryo Fujii; Shuji Hinuma; Naoki Nishizawa; Chieko Kitada; Haruo Onda; Osamu Nishimura; Masahiko Fujino (162-171).
We analyzed the tissue distribution of apelin mRNA in rats by a quantitative reverse transcription-polymerase chain reaction and that of immunoreactive apelin (ir-apelin) by an enzyme immunoassay (EIA) using a monoclonal antibody. The expression levels of apelin mRNA and ir-apelin seemed to be consistent among tissues: they were highly expressed in the lung and mammary gland. By the combination of gel filtration and EIA, we found that the molecular forms of apelin differ among respective tissues: apelin molecules with sizes close to apelin-36 (long forms) were major components in the lung, testis, and uterus, but both long and short (whose sizes were close to [<Glu65]apelin-13) forms were detected in the mammary gland. In Scatchard analyses, the radioiodinated apelin-36 analogue bound to the receptor, APJ, with high affinity. In competitive binding assays, apelin-36 and apelin-19 far more efficiently inhibited the binding of the labeled apelin-36 analogue with APJ than [<Glu65]apelin-13. In analyses for the dissociation of apelin from APJ, unlabeled apelin-36 replaced more rapidly the labeled apelin-36 analogue bound with APJ than [<Glu65]apelin-13. Our results demonstrate that the long and short forms of apelin differently interact with APJ.
Keywords: Apelin; APJ; Distribution; Antibody; Receptor binding;
Three kinds of currents in the canine betaine-GABA transporter BGT-1 expressed in Xenopus laevis oocytes by Greta Forlani; Elena Bossi; Carla Perego; Stefano Giovannardi; Antonio Peres (172-180).
The cloned canine betaine-GABA cotransporter BGT-1 has been heterologously expressed in Xenopus laevis oocytes in order to characterize its electrophysiological properties. Voltage-clamp experiments on transfected oocytes reveal the presence of three types of membrane current which are absent in non-injected oocytes: (i) an organic substrate-independent current (uncoupled current); (ii) a transport-associated current, seen upon addition of betaine or GABA; (iii) presteady-state currents induced by voltage changes. The three kinds of current are analogous to those reported in structurally similar cotransporters. The transport-associated current is strictly dependent on the presence of Na+. The good correlation between the amount of charge underlying the presteady-state currents and the transport-associated current indicates that both processes are due to the activity of the transporter.
Keywords: Amino acid cotransport; Betaine-γ-amino-n-butyric acid transporter-1; Xenopus oocyte;
Role of guanine nucleotides in the regulation of the Ras/cAMP pathway in Saccharomyces cerevisiae by Silvia Rudoni; Sonia Colombo; Paola Coccetti; Enzo Martegani (181-189).
The CDC25 gene product is a guanine nucleotide exchange factor for Ras proteins in yeast. Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction. To test this hypothesis we measured the levels of nucleotides in yeast cells under different growth conditions and the relative amount of Ras2-GTP. The intracellular GTP/GDP ratio was found to be very sensitive to growth conditions: the ratio is high, close to that of ATP/ADP during exponential growth, but it decreases rapidly before the beginning of stationary phase, and it drops further under starvation conditions. The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio. The relative amount of Ras2-GTP changes in a parallel way suggesting that there is a correlation with the cytosolic GTP/GDP ratio. In addition ‘in vitro’ mixed-nucleotide exchange experiments done on purified Ras2 protein demonstrated that the GTP and GDP concentrations influence the extent of Ras2-GTP loading giving further support to their possible regulatory role.
Keywords: Nutrient; Growth regulation; Guanine nucleotide exchange factor; CDC25; Ras; Saccharomyces cerevisiae;
Biphasic cytotoxic mechanism of extracellular ATP on U-937 human histiocytic leukemia cells: involvement of adenosine generation by C Schneider; H Wiendl; A Ogilvie (190-205).
Since extracellular ATP can exhibit cytotoxic activity in vivo and in vitro, its application has been proposed as an alternative anticancer therapy. In this study we investigated the mechanisms of ATP-induced cytotoxicity in a human leukemic cell line (U-937). ATP added as a single dose exceeding 50 μM was cytostatic or even cytotoxic for U-937 cells. Interestingly, growth inhibition by ATP (50–3500 μM) showed a biphasic dose response. Up to 800 μM, ATP was cytotoxic in a dose-dependent manner (EC50 90 μM). In a range between 800 and 2500 μM, cell count was markedly higher despite the higher ATP concentrations. The cytotoxic effect of ATP could be antagonized by addition of uridine as a pyrimidine source and, alternatively, by addition of the nucleoside transmembrane inhibitor dipyridamole. The apoptosis-inducing adenosine A3 receptor was not involved in measurable quantities, since (1) adenosine did not lead to an elevation of intracellular calcium levels, and (2) an unselective A1–3 antagonist (ULS-II-80) could not abrogate the cytotoxic effect. Experiments monitoring extracellular nucleotide metabolism confirmed the assumption that the long-term production and continuous uptake of adenosine, which is extracellularly generated by degradation of ATP, led to an intracellular nucleotide imbalance with pyrimidine starvation. The biphasic dose response to higher ATP concentrations could be explained by the rapid degradation of lower ATP concentrations (300 μM) to adenosine by serum-derived enzymes, whereas higher concentrations (900 μM) only produced small amounts of adenosine due to forward inhibition of AMP hydrolysis by prolonged high ADP levels. FACS analysis revealed that at lower adenosine concentrations (300 μM) a reversible G1 phase arrest of the cell cycle was induced, whereas higher concentrations (1000 μM) triggered apoptosis. Considering ATP as a potential cytostatic drug, our data have important implications concerning metabolic interactions of administered nucleotides.
Keywords: Extracellular adenosine triphosphate; Adenosine; Apoptosis; Nucleotide metabolism; U-937; Leukemic cell;
Analysis of inhibition by H89 of UCP1 gene expression and thermogenesis indicates protein kinase A mediation of β3-adrenergic signalling rather than β3-adrenoceptor antagonism by H89 by J.Magnus Fredriksson; Håkan Thonberg; Kerstin B.E Ohlson; Ken-ichi Ohba; Barbara Cannon; Jan Nedergaard (206-217).
Although it has generally been assumed that protein kinase A (PKA) is essential for brown adipose tissue function, this has not as yet been clearly demonstrated. H89, an inhibitor of PKA, was used here to inhibit PKA activity. In cell extracts, it was confirmed that norepinephrine stimulated PKA activity, which was abolished by H89 treatment. In isolated brown adipocytes, H89 inhibited adrenergically induced thermogenesis (with an IC50 of approx. 40 μM), and in cultured cells, adrenergically stimulated expression of the uncoupling protein-1 (UCP1) gene was abolished by H89 (full inhibition with 50 μM). However, H89 has been reported to be an adrenergic antagonist on β1/β2-adrenoceptors (AR). Although adrenergic stimulation of thermogenesis and UCP1 gene expression are mediated via β3-ARs, it was deemed necessary to investigate whether H89 also had antagonistic potency on β3-ARs. It was found that EC50 values for β3-AR-selective stimulation of cAMP production (with BRL-37344) in brown adipose tissue membrane fractions and in intact cells were not affected by H89. Similarly, the EC50 of adrenergically stimulated oxygen consumption was not affected by H89. As H89 also abolished forskolin-induced UCP1 gene expression, and potentiated selective β3-AR-induced cAMP production, H89 must be active downstream of cAMP. Thus, no antagonism of H89 on β3-ARs could be detected. We conclude that H89 can be used as a pharmacological tool for elucidation of the involvement of PKA in cellular signalling processes regulated via β3-ARs, and that the results are concordant with adrenergic stimulation of thermogenesis and UCP1 gene expression in brown adipocytes being mediated via a PKA-dependent pathway.
Keywords: Protein kinase A; Uncoupling protein-1; Thermogenesis; β3-Adrenoceptor; N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; Brown adipocyte;
Mitogen-activated protein kinase pathway is involved in α6 integrin gene expression in androgen-independent prostate cancer cells: role of proximal Sp1 consensus sequence by Takehisa Onishi; Kensuke Yamakawa; Omar E. Franco; Juichi Kawamura; Masatoshi Watanabe; Taizou Shiraishi; Sohei Kitazawa (218-227).
Metastatic diseases of prostate cancer reveal high expression of α6 integrin and the activation of mitogen-activated protein kinases (MAP kinase). Therefore, the present study was conducted to examine whether MAP kinase pathway is involved in the α6 integrin gene expression in androgen-independent prostate cancer cell lines. α6 integrin mRNA expression, the α6 integrin promoter-induced luciferase activities and MAP kinase enzyme activities in androgen-independent LNCaP and PC-3 cell lines were higher than those in androgen-dependent LNCaP. Deletion and mutation analysis showed that Sp1 consensus sequence at −48 to −43 bp from the transcription start site was necessary for basal promoter activity. Binding of Sp1 to its consensus sequence in three cell lines was confirmed by electrophoretic mobility shift assays. Sp1 binding to its consensus sequence, as well as promoter activity and mRNA expression, were found to be inhibited by an inhibitor of MAP kinase kinase 1 and 2, U0126, in the androgen-independent cell lines. Our results indicate that the proximal Sp1 is necessary for basal promoter activity of the α6 integrin, suggesting that signal transduction from MAP kinases to activation of Sp1 might be involved in α6 integrin gene expression in androgen-independent prostate cancer cell lines.
Keywords: α6 integrin; Sp1; Mitogen-activated protein kinase; Prostate cancer;
Fibroblast growth factors 1 and 2 differently activate MAP kinase in Xenopus oocytes expressing fibroblast growth factor receptors 1 and 4 by Katia Cailliau; Edith Browaeys-Poly; Jean Pierre Vilain (228-233).
The mitogen-activated protein kinase (MAP kinase) signalling cascade activated by fibroblast growth factors (FGF1 and FGF2) was analysed in a model system, Xenopus oocytes, expressing fibroblast growth factor receptors (FGFR1 and FGFR4). Stimulation of FGFR1 by FGF1 or FGF2 and FGFR4 by FGF1 induced a sustained phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) and meiosis reinitiation. In contrast, FGFR4 stimulation by FGF2 induced an early transient activation of ERK2 and no meiosis reinitiation. FGFR4 transduction cascades were differently activated by FGF1 and FGF2. Early phosphorylation of ERK2 was blocked by the dominant negative form of growth factor-bound protein 2 (Grb2) and Ras, for FGF1–FGFR4 and FGF2–FGFR4. The phosphatidylinositol 3-kinase (PI3 kinase) inhibitors wortmannin and LY294002 only prevented the early ERK2 phosphorylation triggered by FGF2–FGFR4 but not by FGF1–FGFR4. ERK2 phosphorylation triggered by FGFR4 depended on the Grb2/Ras pathway and also involved PI3 kinase in a time-dependent manner.
Keywords: Fibroblast growth factor receptor; Mitogen-activated protein kinase; Extracellular signal-regulated protein kinase 2; Phosphatidylinositol 3-kinase; Xenopus laevis oocyte;
Daunorubicin attenuates tumor necrosis factor-α-induced biosynthesis of plasminogen activator inhibitor-1 in human umbilical vein endothelial cells by Shinji Soeda; Kenji Iwata; Yoshiko Hosoda; Hiroshi Shimeno (234-241).
The anthracycline antibiotic daunorubicin is reported to induce apoptosis in cells by triggering ceramide generation through de novo synthesis or sphingomyelin hydrolysis. Treatment of human umbilical vein endothelial cells (HUVEC) with daunorubicin markedly decreased the mRNA expression and protein release of plasminogen activator inhibitor-1 (PAI-1). This cellular event was accompanied by a significant increase in the total ceramide content in HUVEC. On the other hand, tumor necrosis factor (TNF)-α treatment of HUVEC led to an increase in both PAI-1 mRNA expression and protein release, and an enhancement of total ceramide content was also observed. The stimulating effect of TNF-α on PAI-1 synthesis was attenuated by the pretreatment of HUVEC with daunorubicin. Interestingly, the daunorubicin-induced increase in ceramide content was blocked by addition of the potent ceramide synthase inhibitor fumonisin B1, while the TNF-α-induced ceramide increase was not affected by this drug. Fumonisin B1 treatment restored the daunorubicin-induced decrease in PAI-1 release to approximately 70% of the control, but did not affect the TNF-α-induced increase in PAI-1 release. Thus, these data imply the possibility that the subcellular topology of ceramide production determines its lipid mediator function in the regulation of PAI-1 synthesis in HUVEC, because both TNF-α and daunorubicin could increase the ceramide levels.
Keywords: Plasminogen activator inhibitor-1; Daunorubicin; Tumor necrosis factor-α; Ceramide; Vascular endothelial cell;
The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells by Graham P. Feeney; Mark Worwood (242-251).
In order to measure the effects of HFE (haemochromatosis) upon iron uptake, stable expression of wild-type and C282Y, H63D and S65C mutant HFE cDNA was established in HEK 293 cells. Control cells were transfected with empty vector. Expression of HFE mRNA and protein was detected in the cell lines transfected with HFE cDNA, but not in the control cell line. The ferritin concentration in wild-type cells cultured in 40 μM ferric ammonium citrate was 69% of that in control cells and 81% of that in C282Y cells. The ferritin concentration in H63D cells was intermediate between wild-type and C282Y and the ferritin concentration in S65C cells was similar to wild-type cells. Uptake of transferrin-iron in wild-type, C282Y and control cells was measured over 45 min. The Hill coefficients for transferrin-iron uptake were similar. The V max for transferrin-iron uptake in wild-type cells was 59.5% of control cells and 69.5% of C282Y cells. Estimates of K m were 232 nM for wild-type cells, 338 nM for C282Y cells and 570 nM for controls. Transferrin receptor levels were lowered, but not significantly, in the HFE transfected cells. The results show that HFE reduces transferrin-iron uptake, probably as an uncompetitive inhibitor.
Keywords: HFE; Haemochromatosis; Hemochromatosis; Transferrin; Iron;
Receptor-independent effects of natural cannabinoids in rat peritoneal mast cells in vitro by Jean-Luc Bueb; Didier M. Lambert; Eric J. Tschirhart (252-259).
Cannabinoids can activate CB1 and CB2 receptors. Since a CB2 mRNA has been described in rat peritoneal mast cells (RPMC), we investigated a series of cannabinoids and derivatives for their capacity to stimulate RPMC. Effects of natural cannabinoids Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-THC, endocannabinoids (anandamide, palmitoylethanolamide) and related compounds (N-decanoyl-, N-lauroyl-, N-myristoyl-, N-stearoyl- and N-oleoyl-ethanolamines; N-palmitoyl derivatives (-butylamine, -cyclohexylamine, -isopropylamine); and N-palmitoyl, O-palmitoylethanolamine), and synthetic cannabinoids including WIN 55,212-2, SR141716A and SR144528 were assessed for their capacity to induce histamine release or prime RPMC stimulated by compound 48/80. Only Δ9-THC and Δ8-THC could induce non-lytic, energy- and concentration-dependent histamine releases from RPMC (respective EC50 values: 23.5±1.2; 53.4±20.6 μM, and maxima: 71.2±5.5; 55.7±2.7% of the total RPMC histamine content). These were not blocked by CB1 (SR141716A) or CB2 (SR144528) antagonists, but reduced by pertussis toxin (100 ng/ml). Endocannabinoids and analogues did neither induce histamine secretion, nor prime secretion induced by compound 48/80 (0.2 μg/ml). Δ9-THC and Δ8-THC induced in vitro histamine secretion from RPMC through CB receptor-independent interactions, partly involving Gi/o protein activation.
Keywords: Rat peritoneal mast cell; Δ9-Tetrahydrocannabinol; Δ8-Tetrahydrocannabinol; N-Palmitoylethanolamine; Anandamide; Cannabinoid;
LIM domain protein Trip6 has a conserved nuclear export signal, nuclear targeting sequences, and multiple transactivation domains by Yuan Wang; Thomas D. Gilmore (260-272).
Trip6 is a member of a subfamily of LIM domain proteins, including also zyxin, LPP, Ajuba, and Hic-5, which localize primarily to focal adhesion plaques. However, in this report, we demonstrate that Trip6 is largely in the nucleus in cells treated with leptomycin B, suggesting that Trip6 shuttles between nuclear and cytoplasmic compartments and that nuclear export of Trip6 is dependent on Crm1. Consistent with this finding, we have identified a nuclear export signal (NES) in Trip6, and mutation of this NES also results in sequestration of Trip6 in the nucleus. Addition of the Trip6 NES to the nuclear v-Rel oncoprotein redirects v-Rel to the cytoplasm. Trip6 also has at least two sequences that can direct cytoplasmic β-galactosidase to the nucleus. Using GAL4 fusion proteins and reporter gene assays, we demonstrate that Trip6 has multiple transactivation domains, including one that appears to overlap with sequences of the NES. In vitro- or in vivo-synthesized Trip6, however, does not bind to DNA–cellulose. Taken together, these results are consistent with Trip6, and other members of this LIM protein family, having a role in relaying signals between focal adhesion plaques and the nucleus.
Keywords: Trip6; Zyxin; LIM domain; Transcriptional activation; Leptomycin B; Nuclear export signal;
Release of pro- and anti-angiogenic factors by human cardiac fibroblasts: effects on DNA synthesis and protection under hypoxia in human endothelial cells by Lei Zhao; Mahboubeh Eghbali-Webb (273-282).
Myocardium consists of diverse cell types suggesting a role for cell-cell interaction in maintaining the structural and functional integrity of the heart. Cardiac fibroblasts are the source of extracellular matrix, growth factors and cytokines in the heart and their interactions with cardiac myocytes are recognized. Their effects on biological responses of endothelial cells, however, are vastly unexplored. Proliferation of endothelial cells is an essential stage of angiogenesis and contributes to development of coronary collaterals. This study was designed to evaluate the effect of soluble factors produced by cardiac fibroblasts on endothelial cell proliferation. Human cardiac fibroblast-conditioned medium (CF-CM) caused a significant increase (47%, P<0.0001) in DNA synthesis in human umbilical vein endothelial cells (HUVEC), as determined by [3H]thymidine incorporation. This effect was dependent on de novo protein synthesis and activation of MAP kinases. Consistently, CF-CM induced the expression and activation of ERK2 in HUVEC. The CF-CM from which heparin-binding proteins were removed, had a significantly enhanced stimulatory effect on DNA synthesis in HUVEC compared to that of ‘whole CF-CM’. Western analysis showed the presence of VEGF, bFGF, PDGF, TGF-β1, fibronectin and thrombospondin-1 in whole CF-CM. The individual immunodepletion of each factor from whole CF-CM showed that all were necessary for full activity of CF-CM. CF-CM caused a significant reversal of hypoxia-induced inhibition of DNA synthesis and enhanced expression of survival-associated protein, Bcl2, in HUVEC. Together, these data show that cardiac fibroblasts release inhibitory and stimulatory factors, the net effect of which is an enhancement of DNA synthesis in endothelial cells. These results point to the role that cardiac fibroblasts may play in angiogenesis in the heart.
Keywords: Proliferation; Hypoxia; Myocardial ischemia; Angiogenesis; Endothelial cells; Cardiac fibroblasts;
Hyaluronan reduces migration and proliferation in CHO cells by Birgit Dübe; Hans Joachim Lüke; Monique Aumailley; Peter Prehm (283-289).
Expression of the hyaluronan synthase gene in hyaluronan-deficient CHO cells changed the cell morphology from a spindle shape to a flattened epithelial-type form. Hyaluronan producing CHO cells showed reduced initial cell adhesion, migration, proliferation and density at contact inhibition, but no difference in random migration determined by the Boyden chamber assay. Addition of hyaluronan to the medium of CHO cells reduced migration, proliferation and initial cell adhesion. In contrast, coating the plastic dish with hyaluronan enhanced initial cell adhesion. These results are discussed in the context of the perplexing properties of hyaluronan on cellular functions.
Keywords: Hyaluronan; Migration; Proliferation; Adhesion;
Zinc(II)-mediated enhancement of the agonist activity of histidine-substituted parathyroid hormone(1–14) analogues by Percy H Carter; Thomas J Gardella (290-304).
Previous studies on parathyroid hormone (PTH)(1–14) revealed that residues (1–9) played a dominant role in stimulating PTH-1 receptor-mediated increases in cAMP formation. In the present study, we examined the effects of installing a metal-binding motif in the (10–14) region of rat PTH(1–14) on the peptide’s agonist activity. We found that substitution of histidine for the native asparagine at position 10 of PTH(1–14) provided a peptide that was approx. 8-fold more potent as an agonist in the presence of divalent zinc salts than it was in the absence of the metal. This enhancement in potency was dependent on the native histidine at position 14, the concentration of Zn(II) utilized, and did not occur with other divalent metal ions. The zinc-activated [His10]-PTH(1–14) peptide was blocked by a classical PTH-1 receptor antagonist, PTHrP(7–36), and did not activate the PTH-2 receptor. The zinc-mediated enhancing effect did not require the large N-terminal extracellular domain of the PTH-1 receptor. Although we were able to demonstrate that [His10]-PTH(1–14) binds Zn(II) using 1H-NMR, our spectroscopic studies (circular dichroism and nuclear magnetic resonance) were not consistent with the notion that zinc enhanced the activity of [His10]-PTH(1–14) simply by inducing a helical structure in the 10–14 region. Rather, the data suggest that the enhancement in cAMP potency arises from the formation of a ternary complex between [His10]-PTH(1–14), a zinc atom, and the extracellular loop/transmembrane domain region of the PTH-1 receptor.
Keywords: Parathyroid hormone; Peptide hormone; G protein-coupled receptor; Protein engineering; Zinc-binding peptide; Model α-helix;
Ku protein in human T and B lymphocytes: full length functional form and signs of degradation by A Sallmyr; G Henriksson; S Fukushima; A Bredberg (305-312).
DNA-dependent protein kinase (DNA-PK) has been shown to take part in cell cycle regulatory signal transduction and in the repair of X-ray-induced DNA double-strand breaks. Functional DNA-PK is furthermore needed for the generation of antigen specificity during lymphocyte maturation. The Ku86 subunit of DNA-PK has been reported to exist in human B lymphocytes in a truncated form capable of binding to broken DNA but lacking the ability to activate the kinase function of DNA-PK. In the present work the Ku70 and Ku86 dimer proteins in T and B lymphocytes from human blood donors were analysed by immunoblotting and were observed apparently to be of full length. Also, nuclear protein extracted from B and non-B lymphocytes displayed DNA-dependent kinase activity. However, a minor fraction of Ku86 in lymphocytes was observed to be truncated with a molecular mass of approx. 70 kDa.
Keywords: Ku protein; DNA-dependent protein kinase; B lymphocyte; T lymphocyte;
Reduction of phospholemman expression decreases osmosensitive taurine efflux in astrocytes by Julio Morán; Marcela Morales-Mulia; Herminia Pasantes-Morales (313-320).
The role of phospholemman (PLM) in taurine and Cl− efflux elicited by 30% hyposmotic solution was studied in cultured cerebellar astrocytes with reduced PLM expression by antisense oligonucleotide (AO) treatment. PLM, a substrate for protein kinases (PK) C and A, is a protein that increases an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. Two antisense oligonucleotides (AO1 and AO2) effectively decreased the expression of the PLM protein by 40% and 30%, respectively, and markedly reduced [3H]taurine efflux by 67% and 62%. AO treatment also decreased the osmosensitive release of Cl−, followed as 125I. The inhibition of Cl− efflux (23% for AO1 and 13% for AO2) was notably lower than for [3H]taurine. The contribution of PKC and PKA in the function of PLM was also evaluated in astrocytes. Pharmacological activation or inhibition of PKC and PKA revealed that the osmosensitive taurine efflux is essentially PKC-independent while 125I efflux is reduced by the PKC blockers H-7 (21%) and Gö6983 (41%). The PKA activator forskolin and dbcAMP increased taurine efflux by 66–70% and 125I efflux by 21–45%. Norepinephrine increased the osmosensitive taurine efflux at about the same extent as dbcAMP and forskolin, and this was reduced by PKA blockers. These results suggest that PLM plays a role in RVD in astrocytes by predominantly influencing taurine fluxes, which are modulated by PKA but not PKC.
Keywords: Phospholemman; Taurine; Chloride; Volume regulation; Astrocyte; Protein kinase A; Protein kinase C;
Protein oxidation and proteolysis in RAW264.7 macrophages: effects of PMA activation by Jeanette Gieche; Jana Mehlhase; Anke Licht; Thomas Zacke; Nicolle Sitte; Tilman Grune (321-328).
Macrophages are stimulable cells able to increase the production of reactive oxygen and nitrogen species dramatically for a short period of time. Free radicals and other oxidants are able to oxidize the intracellular protein pool. These oxidized proteins are selectively recognized and degraded by the intracellular proteasomal system. We used the mouse macrophage-like cell line RAW264.7 to test whether macrophagial cells are able to increase their protein turnover after oxidative stress and whether this is accompanied by an increased protein oxidation. Macrophagial cells are particularly susceptible to bolus additions of hydrogen peroxide and peroxynitrite. In further experiments we activated RAW264.7 cells with PMA to test whether the production of endogenous oxidants has analogous effects. A clear dependence of the protein turnover and protein oxidation on the oxidative burst could be measured. In further experiments the role of the proteasomal system in the selective removal of oxidized proteins could be revealed exploring the proteasome specific inhibitor lactacystin. Therefore, although oxidants are able to attack the intracellular protein pool in macrophages, these cells are able to remove oxidized proteins selectively and protect the intracellular protein pool from oxidation.
Keywords: Macrophage; Protein oxidation; Proteolysis; Protein degradation; Proteasome;
Hormonal regulation of phospholipase D activity in Ca2+ transporting cells of rabbit connecting tubule and cortical collecting duct by Remko R. Bosch; Joost G.J. Hoenderop; Linda van der Heijden; Jan Joep H.H.M. De Pont; René J.M. Bindels; Peter H.G.M. Willems (329-338).
Phospholipase D (PLD) is distributed widely in mammalian tissues where it is believed to play an important role in the regulation of cell functions and cell fate by a variety of extracellular signals. In this study, we used primary cultures of rabbit connecting tubule (CNT) and cortical collecting duct (CCD) cells, grown to confluence on a permeable support, to investigate the possible involvement of PLD in the mechanism of action of hormones that regulate Ca2+ reabsorption. RT-PCR revealed the presence of transcripts of PLD1b and PLD2, but not PLD1a, in these cultures. Moreover, the expression of substantial amounts of PLD1 protein was demonstrated by Western blotting. To measure PLD activity, cells were labelled with [3H]myristic acid after which the PLD-catalysed formation of radiolabelled phosphatidylethanol ([3H]PtdEth) was measured in the presence of 1% (v/v) ethanol. Deamino-Cys,D-Arg8-vasopressin (dDAVP) and N 6-cyclopentyladenosine (CPA), two potent stimulators of Ca2+ transport across these monolayers, stimulated PLD activity as was indicated by a marked increase in [3H]PtdEth. Similarly, ATP, a potent inhibitor of dDAVP- and CPA-stimulated Ca2+ transport, increased the formation of [3H]PtdEth. PLD activity was furthermore increased by 8Br-cAMP and following acute (30 min) stimulation of protein kinase C (PKC) with a phorbol ester (PMA). Chronic PMA treatment (120 h) to downregulate phorbol ester-sensitive PKC isoforms did not affect PLD activation by dDAVP, CPA and 8Br-cAMP, while markedly decreasing the effect of ATP and abolishing the effect of PMA. The PKC inhibitor chelerythrine significantly reduced PLD activation by dDAVP, CPA and 8Br-cAMP, without changing the effect of ATP. The inhibitor only partially reduced the effect of PMA. This study shows that Ca2+ transporting cells of CNT and CCD contain a regulated PLD activity. The physiological relevance of this activity, which is not involved in Ca2+ reabsorption, remains to be established.
Keywords: Cortical collecting duct; Connecting tubule; Ca2+ reabsorption; Vasopressin; ATP; N 6-Cyclopentyladenosine; Chelerythrine; Phorbol ester;
Molecular analysis of a novel Drosophila diacylglycerol kinase, DGKϵ by Maxim V Frolov; Elizaveta V Benevolenskaya; James A Birchler (339-352).
Diacylglycerol kinase plays a central role in the metabolism of diacylglycerol by converting diacylglycerol into phosphatidic acid thus initiating resynthesis of phosphatidylinositols. Diacylglycerol is a known second messenger reversibly activating protein kinase C. In addition, diacylglycerol is a potential precursor for polyunsaturated fatty acids. We describe the identification and molecular analysis of a novel type III Drosophila diacylglycerol kinase isoform, DGKϵ. Drosophila DGKϵ is mapped to the cytological position 49C1-3. DGKϵ mRNA is 1.9 kb in length and is broadly distributed throughout development in different cells, primordia and organs, including testes. In embryogenesis, the transcripts are enriched in the cells, which are in S-phase or undergoing endoreplication. Comparison of the Drosophila DGKϵ with the human homologue revealed that the first zinc finger-like motif is specific for the type III isoform. Although the testis-specific diacylglycerol kinase activity is dependent upon the dose of DGKϵ gene, the deletion of DGKϵ does not modulate the total cellular diacylglycerol level. In spite of a proposed key role of diacylglycerol kinase in termination of the diacylglycerol signal, overexpression of a DGKϵ transgene in flies under the control of a yeast upstream activating sequence promoter does not disrupt normal development in Drosophila.
Keywords: Drosophila; Diacylglycerol kinase; Oxen locus; Intracellular signaling; Lipid biosynthesis;
Author Index (353-355).
Cumulative Contents (356-357).
Information for Contributors (358-363).