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PubMed2004

Ca(2+)-calmodulin signalling pathway up-regulates GABA synaptic transmission through cytoskeleton-mediated mechanisms.

We investigated the role of calcium (Ca(2+))/calmodulin (CaM) signaling pathways in modulating GABA synaptic transmission at CA1 pyramidal neurons in hippocampal slices. Whole-cell pipettes were used to record type A GABA receptor (GABA(A)R)-gated inhibitory postsynaptic currents (IPSCs) and to perfuse intracellularly modulators in the presence of glutamate receptor antagonists. GABA(A)R-gated IPSCs were enhanced by the postsynaptic infusions of adenophostin (1 microM), a potent agonist of inositol-1,4,5-triphosphate receptor (IP(3)R) that induces Ca(2+) release. The enhancement was blocked by co-infusing either 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (10 mM) or CaM-binding peptide (100 microM). Moreover, the postsynaptic infusion of Ca(2+)-CaM (40/10 microM) enhanced both evoked and spontaneous GABA(A)R-gated IPSCs. The enhancement was attenuated by co-infusing 100 microM CaM-KII(281-301), an autoinhibitory peptide of CaM-dependent protein kinases. These results indicate that postsynaptic Ca(2+)-CaM signaling pathways essentially enhance GABAergic synaptic transmission. In the investigation of synaptic targets for the enhancement, we found that IP(3)R agonist-enhanced GABA(A)R-gated IPSCs were attenuated by co-infusing colchicine (30 microM), vincristine (3 microM) or cytochalasin D (1 microM) that inhibits tubulin or actin polymerization, implying that actin filament and microtubules are involved. We conclude that postsynaptic Ca(2+)-CaM signaling pathways strengthen the function of GABAergic synapses via a cytoskeleton-mediated mechanism, probably the recruitment of receptors in the postsynaptic membrane.

Animals↗
PubMed1996

Induction of apoptosis by diethylstilbestrol in hormone-insensitive prostate cancer cells.

BACKGROUND: Diethylstillbestrol (DES) and diethylstilbestrol diphosphate (DESdP) are effective agents for the treatment of advanced prostate cancers. Tumor-inhibiting effects of DES and DESdP are presumed secondary to suppression of androgen production in vivo. Little is known, however, about the direct cellular mechanisms of the tumor inhibition. Estrogens have been reported not only to stimulate growth but also to disrupt microtubule formation in prostate cancer cells. PURPOSE: The study was designed to examine and compare mechanisms of in vitro growth inhibition of DES and DESdP in human androgen-insensitive prostate cancer cells (DU145, 1-LN, and PC-3) and human androgen-sensitive prostate cancer cells (LNCaP) and to examine estrogen receptor modulation of such effects. METHODS: The cytotoxic effects of DES and DESdP were examined in vitro by use of a standard microculture tetrazolium assay to quantitate numbers of viable cells. Immunofluorescence microscopy, DNA fragmentation analysis, and fluorescence flow cytometry were used to investigate microtubules, the induction of apoptosis, and changes in cell cycle distribution. The degree of estrogen receptor positivity of untreated and treated cells was determined by immunohistochemistry and quantitative image analysis. RESULTS: LD50 levels (the dose at which 50% of cells are no longer viable) in the concentration range of 19-25 microM were observed for both DES and DESdP in all cell lines examined. DESdP-induced growth inhibition was found to be dependent on heat-labile phosphatases present in fetal calf serum. DES-induced cytotoxicity was not affected by the presence of 17 beta-estradiol, and it was not dependent on the presence of estrogen receptor. Estrogen receptor-positive cells and estrogen receptor-negative cells were equally responsive to DES. PC-3 cells stained with fluorescent anti-tubulin, phalloidin (actin stain), and 4',6-diamidino-2-phenylindole (DNA stain) showed no inhibition of microtubules or actin filaments but revealed the presence of apoptotic bodies in the nuclei. Fluorescence flow cytometry of nuclear DNA content of propidium iodide-stained nuclei from androgen-insensitive prostate cancer cells treated with 15 or 30 microM DES or DESdP revealed an increase in relative numbers of hypodiploid (apoptotic) nuclei, a depletion of G1- and S-phase cells, and an accumulation of cells in G2/M phase. Conversely, androgen-sensitive cells contained a lower percentage of hypodiploid nuclei but no accumulation of cells in G2/M phase. CONCLUSIONS: Direct cytotoxic effects of DES in prostate cancer cells are estrogen receptor independent and do not involve disruption of microtubule architecture but do involve the promotion of cell cycle arrest and apoptosis. These are the first data confirming direct cytotoxic effects of DES and DESdP in prostate cancer cells via an apoptotic mechanism. IMPLICATIONS. These results suggest that DES and DESdP have potential value as agents against androgen-insensitive prostate neoplasms through induction of an apoptotic cascade.

Antineoplastic Agents, Hormonal↗
PubMed2004

Overexpression of lunatic fringe does not affect epithelial cell differentiation in the developing mouse lung.

The Notch/Notch-ligand pathway regulates cell fate decisions and patterning in various tissues. Several of its components are expressed in the developing lung, suggesting that this pathway is important for airway cellular patterning. Fringe proteins, which modulate Notch signaling, are crucial for defining morphogenic borders in several organs. Their role in controlling cellular differentiation along anterior-posterior axis of the airways is unknown. Herein, we report the temporal-spatial expression patterns of Lunatic fringe (Lfng) and Notch-regulated basic helix-loop-helix factors, Hes1 and Mash-1, during murine lung development. Lfng was only expressed during early development in epithelial cells lining the larger airways. Those epithelial cells also expressed Hes1, but at later gestation Hes1 expression was confined to epithelium lining the terminal bronchioles. Mash-1 displayed a very characteristic expression pattern. It followed neural crest migration in the early lung, whereas at later stages Mash-1 was expressed in lung neuroendocrine cells. To clarify whether Lfng influences airway cell differentiation, Lfng was overexpressed in distal epithelial cells of the developing mouse lung. Overexpression of Lfng did not affect spatial or temporal expression of Hes1 and Mash-1. Neuroendocrine CGRP and protein gene product 9.5 expression was not altered by Lfng overexpression. Expression of proximal ciliated (beta-tubulin IV), nonciliated (CCSP), and distal epithelial cell (SP-C, T1alpha) markers also was not influenced by Lfng excess. Overexpression of Lfng had no effect on mesenchymal cell marker (alpha-sma, vWF, PECAM-1) expression. Collectively, the data suggest that Lunatic fringe does not play a significant role in determining cell fate in fetal airway epithelium.

Animals↗
PubMed1993

Modulation of antibody-mediated glomerular injury in vivo by interleukin-6.

We have shown previously that pretreatment with small doses of bacterial lipopolysaccharide (LPS), human recombinant interleukin-1 beta (hrIL-1 beta) and human recombinant tumor necrosis factor-alpha (hrTNF) increase injury in the heterologous phase of nephrotoxic nephritis (NTN). All three pretreatments induce synthesis of interleukin-6 (IL-6) which in some systems down-regulates synthesis of IL-1 and TNF. We have now investigated the influence of IL-6 on injury in both heterologous and autologous phases of NTN in rats. Injection of hrIL-6 in doses sufficient to induce hepatic synthesis of acute phase proteins (assessed by plasma alpha 2-macroglobulin concentration) had no effect on glomerular injury in the heterologous phase of NTN (albuminuria in NTAb alone 9 +/- 6; LPS/NTAb 34 +/- 10 and IL-6/NTAb 2 +/- 1 mg/24 hr, P < 0.001, Wilcoxon test). In contrast, IL-6 pretreatment partially abrogated the effect of LPS on albumin excretion (NTAb 4 +/- 2; LPS/NTAb 85 +/- 11 and IL-6/LPS/NTAb 32 +/- 6 mg/24 hr, P < 0.002), percentage of glomerular capillary thrombi (3 +/- 1%; 39 +/- 8%; and 6 +/- 1%, P < 0.001) and glomerular neutrophil infiltrate (29 +/- 3; 58 +/- 5; and 34 +/- 2 neutrophils/50 glomeruli in section, P < 0.001, respectively) at 24 hours. The effect of IL-6 was also evident four hours after induction of nephritis and was associated with a marked reduction in glomerular concentration of mRNA for IL-1 beta and TNF, without change in that of tubulin. Serum TNF concentrations were also significantly reduced at four hours in IL-6 treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Selective and reversible effects of vinca alkaloids on Trypanosoma cruzi epimastigote forms: blockage of cytokinesis without inhibition of the organelle duplication.

Vinca alkaloids, vincristine and vinblastine, produce differential effects on the cell division of Trypanosoma cruzi epimastigote forms depending on drug concentrations. These effects are related to different microtubule-based mechanisms. For 15 microM vinblastine and 50 microM vincristine, the drugs inhibit both nuclear division and cytokinesis, and affect cell shape. At 3 microM vinblastine and 10 microM vincristine, however, cytokinesis is inhibited without major effect on the progression of the cell cycle; this yields giant cells having multiple nuclei, kinetoplasts and flagella. Cultures maintained over 1 week with daily drug replacement produced cells with more than 16 nuclei and 24 kinetoplasts, indicating that an equivalent of a fifth cell cycle was initiated. The ultrastructure of the multinucleate cells showed a basic organization closely similar to that of trypanosomes. Cytokinesis inhibition by vinca alkaloids seems to result from modulations of interactions between microtubules and associated proteins, rather than from an inhibition of microtubule dynamics as is usually proposed for vinca alkaloids. Cytokinesis inhibition is reversible: after removing the drug, epimastigotes emerge from the multinucleate cells. The emerging process follows a precise axis and polarity which are determined by the position of the flagellum/kinetoplast complex. This region could play an essential role in cell morphogenesis since zoids (cells without a nucleus) are frequently observed.

Animals↗
PubMed2005

The transmembrane semaphorin Sema6A controls cerebellar granule cell migration.

The transmembrane semaphorin protein Sema6A is broadly expressed in the developing nervous system. Sema6A repels several classes of developing axons in vitro and contributes to thalamocortical axon guidance in vivo. Here we show that during cerebellum development, Sema6A is selectively expressed by postmitotic granule cells during their tangential migration in the deep external granule cell layer, but not during their radial migration. In Sema6A-deficient mice, many granule cells remain ectopic in the molecular layer where they differentiate and are contacted by mossy fibers. The analysis of ectopic granule cell morphology in Sema6a-/- mice, and of granule cell migration and neurite outgrowth in cerebellar explants, suggests that Sema6A controls the initiation of granule cell radial migration, probably through a modulation of nuclear and/or soma translocation. Finally, the analysis of mouse chimeras suggests that this function of Sema6A is primarily non-cell-autonomous.

Analysis of Variance↗
PubMed2002

Enhanced microtubule-dependent trafficking and p53 nuclear accumulation by suppression of microtubule dynamics.

The tumor suppressor protein p53 localizes to microtubules (MT) and, in response to DNA damage, is transported to the nucleus via the MT minus-end-directed motor protein dynein. Dynein is also responsible for MT-mediated nuclear targeting of adenovirus type 2 (Ad2). Here we show that treatment with low concentrations of MT-targeting compounds (MTCs) that do not disrupt the MT network but are known to suppress MT dynamics enhanced p53 nuclear accumulation, and the activation of the p53-downstream target genes. p53 nuclear accumulation required binding of MTCs to MTs and enhanced the induction of p53-up-regulated modulator of apoptosis (PUMA) mRNA and apoptosis on challenging cells with the DNA-damaging drug adriamycin. Low concentrations of MTCs enhanced the rate of movement of fluorescent Ad2 to the nucleus and increased the nuclear targeting efficiency of Ad2. We propose that suppression of MT dynamics by low concentrations of MTCs enhances MT-dependent trafficking toward the minus ends of MTs and facilitates nuclear targeting.

Adenoviruses, Human↗
PubMed1997

The cytotoxicity of the parvovirus minute virus of mice nonstructural protein NS1 is related to changes in the synthesis and phosphorylation of cell proteins.

Autonomous parvoviruses exert lytic and cytostatic effects believed to contribute to their antineoplastic activity. Studies with inducible clones have demonstrated a direct involvement of parvovirus nonstructural proteins (NS) in oncolysis. Human and rat fibroblasts have been stably transfected with MVM(p) (minute virus of mice prototype strain) NS genes cloned under the control of a hormone-inducible promoter. Dexamethasone-induced synthesis of the NS proteins in sensitive transformed cells results in cell killing within a few days. From these sensitive cell lines have been isolated some NS-resistant clones that also prove resistant to MVM(p) infection, suggesting that cell factors modulate NS cytotoxicity. We have previously reported that factors involved in cell cycle regulation may contribute to this modulation, since NS toxicity requires cell proliferation and correlates with a cell cycle perturbation leading to an arrest in phase S/G2. In addition to its role in cytotoxicity, NS1 can regulate transcription driven by parvovirus and nonparvovirus promoters. Since phosphorylation is a critical event in controlling the activity of many proteins, notably transcription factors and cell cycle-regulated proteins, we have examined the effect of NS1 on the synthesis and phosphorylation of cell proteins. Our results indicate that NS1 interferes, within 7 h of induction, with phosphorylation of a protein of about 14 kDa (p14). Cell synchronization has enabled us to show that phosphorylation of this protein occurs in early S phase and is prevented when NS1 is induced. This early effect of NS1 on p14 phosphorylation may be directly linked to cytotoxicity and is probably related to the previously reported inhibition of cell DNA synthesis. Late in the induction period (24 h), NS1 also alters the synthesis of a 50-kDa protein and a 35-kDa protein (p50 and p35, respectively). Microsequencing of p35 reveals sequence homology with beta-tubulin. These effects of NS1, observed only in NS1-sensitive cell lines, may be related to the protein's cytotoxicity.

Amino Acid Sequence↗
PubMed2002

Effect of hyperthermia on the transport of mRNA encoding the extracellular matrix glycoprotein SC1 into Bergmann glial cell processes.

SC1 is an extracellular matrix glycoprotein that is related to the multifunctional protein SPARC. These matricellular members play regulatory roles in modulating cellular interactions. SC1 expression is enriched in the central nervous system during embryonic and postnatal development as well as in the adult brain. In the rat cerebellum, SC1 is expressed at high levels in Bergmann glial cells and their radial fibers which project into the synaptic-rich molecular layer. At specific stages of development and in the adult, SC1 mRNA is selectively transported into cellular processes of these cells. In the present study, we have examined the effect of whole-body hyperthermia on the transport of SC1 mRNA in Bergmann glial cells of the rat cerebellum. Our results show that SC1 mRNA transport is diminished at 10 and 15 h post-hyperthermia, but returns to control levels by 24 h after heat shock. One of the characteristics of a heat shock on cells grown in tissue culture is a collapse of the cytoskeletal network. Intact components of the cytoskeleton are necessary for the transport of mRNA into peripheral processes of cells. However, in vivo hyperthermia does not appear to affect the morphology of the intermediate filament proteins GFAP, vimentin, or the beta-tubulin component of microtubules in Bergmann glial cell processes. During the hyperthermic time course, levels of vimentin protein increase, which is reflected by immunoreactivity of activated astrocytes and microvasculature in cerebellar white matter.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1996

Regional differences in in vitro growth of neural cell processes during development.

Primary cell cultures from cerebral cortex, striatum and ventral mesencephalon obtained from rat fetal (embryonic day 17, E17) or postnatal (day 2, PN2) donors were grown either in media conditioned by subcultured astroglia from the same regions, an artificial trophic medium, normal human amniotic fluid, or in normal human cerebrospinal fluid. To estimate the presence of neuronal-like and non-neuronal cells, cell morphology and immunocytochemistry against microtubule-associated proteins and beta-tubulin were taken into consideration. The percentage of emitting neural cells and length of cell processes were determined after 24 hr in culture. Growth of cell processes in neuronal and non-neuronal cells from prenatal striatum was minimal compared with that in cerebral cortex and ventral mesencephalon, regardless of the culture condition. Nerve growth factor, basic fibroblast growth factor or epidermal growth factor did not significantly modify cell growth in E17 cultures, except for epidermal growth factor, which reduced the number of emitting cells in striatal cultures and increased it in cerebral cortex ones. Cultures derived from postnatal striatum showed a significant increase in neurite length when grown in an astroglial conditioned medium as compared to cultures derived from prenatal (E17) striatum. Results suggest significant regional differences in the brain regarding growth of cell processes at age E17, and reversal of striatal ability to grow cell processes by postnatal day 2. Reduced growth of cell processes showed by E17 striatum cultures was rather independent of the culture media. This fact could suggest that such early regional differences would depend on characteristics of sublineages present at this developmental stage, which would modulate the organization of regional neuropils. The restricted growth of cell processes in cultures from E17 striatum, no longer present in postnatal striatum, suggests that inputs to the striatum may modify expression of cell lineages at later stages of development.

Amniotic Fluid↗
PubMed1988

Transforming growth factor-beta increases transcription of the genes encoding the epidermal growth factor receptor and fibronectin in normal rat kidney fibroblasts.

Transforming growth factor-beta (TGF-beta) is a potent modulator of cell growth in many systems. In normal rat kidney (NRK) fibroblasts, TGF-beta synergizes with epidermal growth factor (EGF) to stimulate growth in soft agar, a characteristic of the transformed phenotype. Many biochemical effects of TGF-beta occur at the cell surface. Increased binding of EGF and synthesis of extracellular matrix components such as fibronectin and collagen are primary responses of NRK cells to TGF-beta. Although specific membrane receptors for TGF-beta have been identified, the mechanism of action of this factor is not well understood. Here we demonstrate that TGF-beta enhances the expression of the EGF receptor in NRK cells through an increase in the level of EGF receptor gene transcripts. Analysis of nuclear run-off transcription levels and mRNA half-lives indicate that the elevation in EGF-receptor mRNA results from an increase in the rate of transcription. Dose-response and kinetic studies suggest that the EGF receptor response to TGF-beta is biphasic, possibly resulting from the action of multiple TGF-beta receptors. TGF-beta also elevates the levels of fibronectin and tubulin transcripts in NRK cells; however, the mechanism differs for each gene. The increase in fibronectin mRNA in response to TGF-beta results from an increased rate of gene transcription. Tubulin mRNA levels, in contrast, appear to be post-transcriptionally regulated. These results implicate TGF-beta as a transcriptional activator of the genes for both the EGF receptor and fibronectin and suggest the two genes may be regulated through a common pathway in this cell type.

Animals↗
PubMed1998

Targeting, capture, and stabilization of microtubules at early focal adhesions.

By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a "cross talk" between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.

3T3 Cells↗
PubMed1997

Distribution and activation of intracellular Ca2+ stores in cultured olfactory bulb neurons.

The presence and distribution of intracellular Ca2+ release pathways in olfactory bulb neurons were studied in dissociated cell cultures. Histochemical techniques and imaging of Ca2+ fluxes were used to identify two major intracellular Ca2+ release mechanisms: inositol 1, 4,5-triphosphate receptor (IP3R)-mediated release, and ryanodine receptor-mediated release. Cultured neurons were identified by immunocytochemistry for the neuron-specificmarker beta-tubulin III. Morphometric analyses and immunocytochemistry for glutamic acid-decarboxylase revealed a heterogeneous population of cultured neurons with phenotypes corresponding to both projection (mitral/tufted) and intrinsic (periglomerular/granule) neurons of the in vivo olfactory bulb. Immunocytochemistry for the IP3R, and labeling with fluorescent-tagged ryanodine, revealed that, irrespective of cell type, almost all cultured neurons express IP3R and ryanodine binding sites in both somata and dendrites. Functional imaging revealed that intracellular Ca2+ fluxes can be generated in the absence of external Ca2+, using agonists specific to each of the intracellular release pathways. Local pressure application of glutamate or quisqualate evoked Ca2+ fluxes in both somata and dendrites in nominally Ca2+ free extracellular solutions, suggesting the presence of IP3-dependent Ca2+ release. These fluxes were blocked by preincubation with thapsigargin and persisted in the presence of the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Local application of caffeine, a ryanodine receptor agonist, also evoked intracellular Ca2+ fluxes in the absence of extracellular Ca2+. These Ca2+ fluxes were suppressed by preincubation with ryanodine. In all neurons, both IP3- and ryanodine-dependent release pathways coexisted, suggesting that they interact to modulate intracellular Ca2+ concentrations.

Animals↗
PubMed1993

Characterization of a PC12 cell sub-clone (PC12-C41) with enhanced neurite outgrowth capacity: implications for a modulatory role of high molecular weight tau in neuritogenesis.

To address the means by which diversity of neuronal morphology is generated, we have isolated and characterized naturally occurring variants of rat PC12 pheochromocytoma cells that exhibit altered neurite outgrowth properties in response to nerve growth factor (NGF). We describe here a PC12 cell sub-clone, designated PC12-clone 41 (PC12-C41), that displays significant increases in neurite abundance and stability when compared with the parental line. This difference does not appear to be due to an altered sensitivity or responsiveness to NGF or to a more rapid rate of neurite extension. Because of the role of the cytoskeleton in neuritogenesis, we examined a panel of the major cytoskeletal proteins (MAP 1.2/1B, beta-tubulin, chartins, peripherin, and high and low molecular weight (HMW and LMW) taus) whose levels and/or extent of phosphorylation are regulated by NGF in PC12 cultures. Although most cytoskeletal proteins showed little difference between PC12 and PC12-C41 cells (+/- NGF treatment), there was a significant contrast between the two lines with respect to tau expression. In particular, while NGF increases the total specific levels of tau in both cell types to similar extents (by about twofold), the proportion comprising HMW tau is threefold higher in the PC12-C41 clone than in PC12 cells. A comparable difference was observed under substratum conditions that were non-permissive for neurite outgrowth and so this effect was not merely a consequence of the differential neuritogenic capacities of the two lines. The distinction between the expression of HMW and LMW taus in PC12 and PC12-C41 cells (+/- NGF) was also observed at the level of the messages encoding these proteins. Such findings indicate that initiation of neurite outgrowth in PC12 cultures does not require a massive induction of tau expression and raise the possibility that HMW and LMW taus may have differential capacities for modulating neuronal morphology.

Animals↗
PubMed2006

Microtubule protofilament number is modulated in a stepwise fashion by the charge density of an enveloping layer.

Microtubules are able to adjust their protofilament (PF) number and, as a consequence, their dynamics and function, to the assembly conditions and presence of cofactors. However, the principle behind such variations is poorly understood. Using synchrotron x-ray scattering and transmission electron microscopy, we studied how charged membranes, which under certain conditions can envelop pre-assembled MTs, regulate the PF number of those MTs. We show that the mean PF number, , is modulated primarily by the charge density of the membranes. decreases in a stepwise fashion with increasing membrane charge density. does not depend on the membrane-protein stoichiometry or the solution ionic strength. We studied the effect of taxol and found that increases logarithmically with taxol/tubulin stoichiometry. We present a theoretical model, which by balancing the electrostatic and elastic interactions in the system accounts for the trends in our findings and reveals an effective MT bending stiffness of order 10-100 k(B)T/nm, associated with the observed changes in PF number.

Animals↗
PubMed1986

Multiple hormone-inducible enhancers as mediators of differential transcription.

Sets of genes under a common regulatory control in a given cell type are often differentially transcribed. The possibility that this differential transcription can be modulated by the number or strength of cis-acting regulatory sequences associated with a given gene was tested by using the glucocorticoid-responsive enhancer element associated with the mouse mammary tumor virus promoter. Results indicate that differential levels of hormone-inducible gene expression can be modulated in an additive way by the number of glucocorticoid-responsive enhancers associated with this promoter. Realization of these effects shows little preference for position of the additional elements with respect to the promoter. When sequences that bind the glucocorticoid receptor in vitro with somewhat lower affinity than the enhancer were tested, these additive effects were not detected. The results support that differential transcription of genes subject to a common regulatory control can be mediated, at least in part, by the number or strength of their associated cis-acting regulatory sequences.

Acetyltransferases↗
PubMed2005

Identification of novel protein targets for modification by 15-deoxy-Delta12,14-prostaglandin J2 in mesangial cells reveals multiple interactions with the cytoskeleton.

The cyclopentenone prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) has been shown to display protective effects against renal injury or inflammation. In cultured mesangial cells (MC), 15d-PGJ2 inhibits the expression of proinflammatory genes and modulates cell proliferation. Therefore, cyclopentenone prostaglandins (cyPG) have been envisaged as a promise in the treatment of renal disease. The effects of 15d-PGJ2 may be dependent on or independent from its role as a peroxisome proliferator-activated receptor agonist. It was shown recently that an important determinant for the peroxisome proliferator-activated receptor-independent effects of 15d-PGJ2 is the capacity to modify proteins covalently and alter their function. However, a limited number of protein targets have been identified to date. Herein is shown that a biotinylated derivative of 15d-PGJ2 recapitulates the effects of 15d-PGJ2 on the stress response and inhibition of inducible nitric oxide synthase levels and forms stable adducts with proteins in intact MC. Biotinylated 15d-PGJ2 was then used to identify proteins that potentially are involved in cyPG biologic effects. Extracts from biotinylated 15d-PGJ2-treated MC were separated by two-dimensional electrophoresis, and the spots of interest were analyzed by mass spectrometry. Identified targets include proteins that are regulated by oxidative stress, such as heat-shock protein 90 and nucleoside diphosphate kinase, as well as proteins that are involved in cytoskeletal organization, such as actin, tubulin, vimentin, and tropomyosin. Biotinylated 15d-PGJ2 binding to several targets was confirmed by avidin pull-down. Consistent with these findings, 15d-PGJ2 induced early reorganization of vimentin and tubulin in MC. The cyclopentenone moiety and the presence of cysteine were important for vimentin rearrangement. These studies may contribute to the understanding of the mechanism of action and therapeutic potential of cyPG.

Amino Acid Sequence↗
PubMed2004

Neural stem cells express melatonin receptors and neurotrophic factors: colocalization of the MT1 receptor with neuronal and glial markers.

BACKGROUND: In order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different in vitro conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT1 and MT2 receptors are expressed in NSCs. RESULTS: RT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT1 receptor mRNA was detected in NSCs maintained for two days in culture, but the MT2 receptor was not seen. An immature MT1 receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 - 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT1 receptor is expressed in both neural (beta-tubulin III positive) and glial (GFAP positive) progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours. CONCLUSIONS: The phenotypic characteristics of C17.2 cells suggest that they are a heterogeneous population of NSCs including both neural and glial progenitors, as observed under the cell culture conditions used in this study. These NSCs have an intrinsic ability to express neurotrophic factors, with an apparent suppression of GDNF expression after several days in culture. The detection of melatonin receptors in neural stem/progenitor cells suggests involvement of this pleiotropic hormone in mammalian neurodevelopment. Moreover, the ability of melatonin to induce GDNF expression in C17.2 cells supports a functional role for the MT1 receptor expressed in these NSCs. In view of the potency of GDNF in promoting the survival of dopaminergic neurons, these novel findings have implications for the utilization of melatonin in neuroprotective strategies, especially in Parkinson's disease.

Animals↗