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PubMed2003

Compartmentation of metabolism probed by [2-13C]alanine: improved 13C NMR sensitivity using a CryoProbe detects evidence of a glial metabolon.

The labelling of metabolites with the NMR active nucleus 13C allows not only metabolite enrichments to be monitored, but also the relative fluxes through competing pathways to be delineated. [2-13C, 15N]alanine was used as a metabolic probe to investigate compartmentation in superfused cerebral slices. Perchloric acid extracts of the tissue were investigated using 13C NMR spectroscopy. The spectra were obtained using a CryoProbe optimised for 13C detection (dual CryoProbe [13C, 1H]) in which the receiver and transmitter coils are cooled to approximately 20K to reduce contributions to noise in the signal obtained. Compared with conventional inverse geometry probe, the signal-to-noise ratio (S/N) was increased by approximately 17-fold using this device. A large proportion of alanine was initially metabolised over the first 20 min by glial cells, as indicated by the relative importance of the glial, only enzyme pyruvate carboxylase to the labelling pattern of glutamate, with the ratio of pyruvate carboxylase to pyruvate dehydrogenase derived glutamate being 0.25, and exported [2-13C, 15N]aspartate. Using the increased sensitivity of the CryoProbe, [2-13C, 15N]aspartate was also detected in the extracts of cerebral tissue. This metabolite could only have been derived via the pyruvate carboxylase pathway, and given the large export of the metabolite into the superfusion buffer suggests the occurrence of a "metabolon" arrangement of enzymes within glial cells.

Alanine↗
PubMed2004

Somatodendritic localization and mRNA association of the splicing regulatory protein Sam68 in the hippocampus and cortex.

The RNA-binding protein Sam68 has been implicated in the signal-dependent processing of pre-mRNA and in the utilization of intron-containing retroviral mRNAs. Sam68 is predominantly nuclear but exhibits remarkable binding affinity for signalling proteins located at the membrane. We have investigated the subcellular distribution of Sam68 in adult rat cortex and hippocampus. Subcellular fractionation showed that the protein was most abundant in nuclei but also was present at a significant level in the cytosol and membrane fractions, including light and synaptic membranes derived from crude synaptosomes. Sam68 extracted from the synaptosomal fraction cosedimented with polysomes on sucrose gradients. In agreement with these findings, immunohistochemical staining indicated that Sam68 was concentrated in neuronal nuclei but was also detectable in the soma and dendrites. Sam68 immunoreactivity examined at the ultrastructural level was found to associate with dendritic microtubules, endoplasmic reticulum, and free polyribosomes, sometimes close to synapses. A combination of immunoprecipitation and RT-PCR directly confirmed that Sam68 was bound to polyadenylated mRNA in cortical lysates. The alphaCaMKII mRNA was identified as one of the coprecipitated transcripts; in contrast, the gephyrin and NR1-1 mRNAs were not coprecipitated, indicating a certain degree of sequence specificity in the association. In electrophoretic mobility shift assays, recombinant GST-Sam68 as well as brain-derived Sam68 bound with high affinity to the alphaCaMKII 3' untranslated region. These results suggest that Sam68 may accompany and, conceivably, regulate mature mRNAs during nuclear export, somatodendritic transport, and translation.

Animals↗
PubMed2006

Jab1 mediates cytoplasmic localization and degradation of West Nile virus capsid protein.

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G(2) phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.

Active Transport, Cell Nucleus↗
PubMed2004

Depolarization-induced translocation of the RNA-binding protein Sam68 to the dendrites of hippocampal neurons.

The traffic and expression of mRNAs in neurons are modulated by changes in neuronal activity. The regulation of neuronal RNA-binding proteins is therefore currently receiving attention. Sam68 is a ubiquitous nuclear RNA-binding protein implicated in post-transcriptional processes such as signal-dependent splice site selection. We show that Sam68 undergoes activity-responsive translocation to the soma and dendrites of hippocampal neurons in primary culture. In unstimulated neurons transiently expressing a GFP-Sam68 fusion protein, 90% of the cells accumulated the protein exclusively in the nucleus, and 4% showed extension of GFP-Sam68 to the dendrites. This nuclear expression pattern required the integrity of the Sam68 N-terminus. When present, the dendritic GFP-Sam68 formed granules, 26% of which were colocalized with ethidium bromide-stained RNA clusters. Most of the GFP-Sam68 granules were completely stationary, but a few moved in either a retrograde or anterograde direction. Following depolarization by 25 mM KCl, 50% of neurons displayed dendritic GFP-Sam68. GFP-Sam68 invaded the dendrites after 2 hours with high KCl, and returned to the nucleus within 3 hours after termination of the KCl treatment. A control GFP fusion derived from the SC-35 splicing factor remained fully nuclear during depolarization. No significant change was observed in the phosphorylation of Sam68 after depolarization. Translocation of Sam68 to the distal dendrites was microtubule dependent. Blockade of calcium channels with nimodipine abolished the translocation. Furthermore, inhibition of CRM-1-mediated nuclear export by leptomycin B partially prevented the depolarization-induced nuclear efflux of GFP-Sam68. These results support the possible involvement of Sam68 in the activity-dependent regulation of dendritic mRNAs.

Active Transport, Cell Nucleus↗
PubMed2004

Structure of a knockout mutant of influenza virus M1 protein that has altered activities in membrane binding, oligomerisation and binding to NEP (NS2).

The influenza virus M1 (matrix) protein forms the connection between the membrane component of the virus and its replication component eight ribonucleoprotein particles (RNPs). For this activity, M1 self-polymerises in the infected cell in order to pull glycoprotein containing membrane segments together. Later in the process of infection, M1 enters the nucleus and is active in the nuclear export process of newly made RNPs for virus particle assembly. The N-terminal domain (residues 1-164) of M1 carries the nuclear localisation sequence (NLS) motif and is important for membrane binding, self-polymerisation and nuclear export of RNPs. An NLS-mutant M1 has been used in functional studies in order to implicate the positive charges in the NLS in these three activities. In this paper, the crystal structure of the N-terminal domain of this NLS-mutant is determined and is found to be the same as that of the wild-type protein, clearly indicating that it is the absence of the positively charged residues of the NLS that causes the knock-out phenotype rather than a change in the overall structure of the mutant protein.

Active Transport, Cell Nucleus↗
PubMed2005

Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506.

Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membrane-based steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.

Animals↗
PubMed2002

Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein.

The core oscillator generating circadian rhythms in eukaryotes is composed of transcription--translation-based autoregulatory feedback loops in which clock gene products negatively affect their own expression. A key step in this mechanism involves the periodic nuclear accumulation of clock proteins following their mRNA rhythms after approximately 6 h delay. Nuclear accumulation of mPER2 is promoted by mCRY proteins. Here, using COS7 cells and mCry1/mCry2 double mutant mouse embryonic fibroblasts transiently expressing GFP-tagged (mutant) mPER2, we show that the protein shuttles between nucleus and cytoplasm using functional nuclear localization and nuclear export sequences. Moreover, we provide evidence that mCRY proteins prevent ubiquitylation of mPER2 and subsequent degradation of the latter protein by the proteasome system. Interestingly, mPER2 in turn prevents ubiquitylation and degradation of mCRY proteins. On the basis of these data we propose a model in which shuttling mPER2 is ubiquitylated and degraded by the proteasome unless it is retained in the nucleus by mCRY proteins.

3T3 Cells↗
PubMed2005

Molecular mechanism of inhibition of nonclassical FGF-1 export.

Fibroblast growth factor (FGF-1) lacks a signal sequence and is exported by an unconventional release mechanism. The nonclassical export of FGF-1 has been shown to be inhibited by an anti-allergic and anti-inflammatory drug, amlexanox (AMX). We investigate the molecular mechanism(s) underlying the inhibitory action of AMX on the release of FGF-1, using a variety of biophysical techniques including multidimensional NMR spectroscopy. AMX binds to FGF-1 and enhances its conformational stability. AMX binds to locations close to Cys30 and sterically blocks Cu(2+)-induced oxidation, leading to the formation of the homodimer of FGF-1. AMX-induced inhibition of the formation of the FGF-1 homodimer is observed both under cell-free conditions and in living cells. Results of this study suggest a novel approach for the design of drugs against FGF-1-mediated disorders.

Aminopyridines↗
PubMed2000

A novel shuttle protein binds to RNA helicase A and activates the retroviral constitutive transport element.

The constitutive transport element (CTE) of type D retroviruses mediates the nuclear export of unspliced viral transcripts. We previously showed that RNA helicase A functionally interacts with CTE and contains a bidirectional nuclear transport domain at the carboxyl terminus. Here we report the identification of a novel human protein, helicase A-binding protein 95 (HAP95), which specifically binds to the carboxyl terminus of RNA helicase A. HAP95 is partially homologous to AKAP95, a member of the A kinase-anchoring protein family, but lacks the protein kinase A binding domain characteristic of this family. HAP95 is a nuclear protein at steady state but shuttles between the nucleus and cytoplasm. Overexpression of HAP95 significantly increases CTE-dependent gene expression but has no effect on general gene expression or that mediated by the Rev/Rev response element of human immunodeficiency virus type 1.

Amino Acid Sequence↗
PubMed2003

Caspase-3-mediated cleavage of Cdc6 induces nuclear localization of p49-truncated Cdc6 and apoptosis.

We show that Cdc6, an essential initiation factor for DNA replication, undergoes caspase-3-mediated cleavage in the early stages of apoptosis in HeLa cells and SK-HEP-1 cells induced by etoposide, paclitaxel, ginsenoside Rh2, or tumor necrosis factor-related apoptosis-inducing ligand. The cleavage occurs at the SEVD442/G motif and generates an N-terminal truncated Cdc6 fragment (p49-tCdc6) that lacks the carboxy-terminal nuclear export sequence. Cdc6 is known to be phosphorylated by cyclin A-cyclin dependent kinase 2 (Cdk2), an event that promotes its exit from the nucleus and probably blocks it from initiating inappropriate DNA replication. In contrast, p49-tCdc6 translocation to the cytoplasm is markedly reduced under the up-regulated conditions of Cdk2 activity, which is possibly due to the loss of nuclear export sequence. Thus, truncation of Cdc6 results in an increased nuclear retention of p49-tCdc6 that could act as a dominant negative inhibitor of DNA replication and its accumulation in the nucleus could promote apoptosis. Supporting this is that the ectopic expression of p49-tCdc6 not only promotes apoptosis of etoposide-induced HeLa cells but also induces apoptosis in untreated cells. Thus, the caspase-mediated cleavage of Cdc6 creates a truncated Cdc6 fragment that is retained in the nucleus and induces apoptosis.

Active Transport, Cell Nucleus↗
PubMed2001

A gamma-2 herpesvirus nucleocytoplasmic shuttle protein interacts with importin alpha 1 and alpha 5.

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus. This is an increasing important subfamily of herpesviruses due to the identification of the first human gamma-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS open reading frame (ORF) 57 protein is a multifunctional trans-regulatory protein homologous to genes identified in all classes of herpesviruses. Recent analysis has demonstrated that ORF 57 has the ability to bind viral RNA and to shuttle between the nucleus and cytoplasm, and is required for efficient nuclear export of viral transcripts. Here we have investigated the nucleocytoplasmic shuttling mechanism utilized by the ORF 57 protein. The yeast two-hybrid system was employed to identify interacting cellular proteins using ORF 57 as bait. We demonstrate that ORF 57 interacts with importin alpha isoforms 1 and 5. In addition, the binding of ORF 57 to importin alpha was mediated by the importin alpha hydrophobic internal armadillo repeats. An ORF 57 amino-terminal arginine-rich sequence, which functions as a nuclear localization sequence, was also required for this interaction. Furthermore, the ORF 57 protein is responsible for the redistribution of importin alpha into the nucleoli. These results identify novel cellular interactions essential for the functioning of this important herpesvirus regulatory protein.

Amino Acid Sequence↗
PubMed2004

Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shock.

Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5 degrees C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37 degrees C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair.

Acid Anhydride Hydrolases↗
PubMed1997

Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants.

DNA-Binding Proteins↗
PubMed2003

Sam68 enhances the cytoplasmic utilization of intron-containing RNA and is functionally regulated by the nuclear kinase Sik/BRK.

Cells normally restrict the nuclear export and expression of intron-containing mRNA. In many cell lines, this restriction can be overcome by inclusion of cis-acting elements, such as the Mason-Pfizer monkey virus constitutive transport element (CTE), in the RNA. In contrast, we observed that CTE-mediated expression from human immunodeficiency virus Gag-Pol reporters was very inefficient in 293 and 293T cells. However, addition of Sam68 led to a dramatic increase in the amount of Gag-Pol proteins produced in these cells. Enhancement of CTE function was not seen when a Sam68 point mutant (G178E) that is defective for RNA binding was used. Additionally, the effect of Sam68 was inhibited in a dose-dependent manner by coexpression of an activated form of the nuclear kinase Sik/BRK that hyperphosphorylated Sam68. RNA analysis showed that cytoplasmic Gag-Pol-CTE RNA levels were only slightly enhanced by the addition of Sam68, compared to a 60- to 70-fold increase in the levels of Gag-Pol protein expression. Thus, in this system, Sam68 functioned to enhance the cytoplasmic utilization of RNA containing the CTE. These results suggest that Sam68 may interact with specific RNAs in the nucleus to provide a "mark" that affects their cytoplasmic fate. They also provide further evidence of links between signal transduction and RNA utilization.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Regulatory role of PI 3-kinase on expression of Cdk4 and p27, nuclear localization of Cdk4, and phosphorylation of p27 in corneal endothelial cells.

PURPOSE: FGF-2 is a potent mitogen of rabbit corneal endothelial cells (CECs). This study was undertaken to investigate whether PI 3-kinase participates in cell cycle regulation in response to stimulation with FGF-2 in CECs. METHODS: Cell proliferation was assayed by counting the cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of cyclin-dependent kinase 4 (Cdk4), p27(Kip1) (p27), phosphatidylinositol 3 (PI 3)-kinase, protein kinase B/Akt (Akt), and beta-actin was analyzed by immunoblot. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. LY294002 was used to inhibit PI 3-kinase. RESULTS: CEC required prolonged and continuous exposure to FGF-2. FGF-2 at 10 ng/mL markedly stimulated PI 3-kinase enzyme activity, and stimulation with FGF-2 also caused activation of Akt. LY294002 inhibited both cell proliferation and PI 3-kinase activity in a concentration-dependent manner. The role of PI 3-kinase in cell cycle stimulation was determined: FGF-2 markedly upregulated expression of Cdk4 and stimulated translocation of Cdk4 into nuclei, whereas LY294002 markedly blocked upregulation of Cdk4 expression, and the inhibitor facilitated nuclear export of Cdk4. In contrast, FGF-2 significantly downregulated expression of p27 and facilitated phosphorylation of p27. LY294002 completely blocked the action of FGF-2 on the expression and phosphorylation of p27. CONCLUSIONS: These data indicate that PI 3-kinase ultimately leads to activation of the cell cycle machinery in response to FGF-2. It does so by upregulating expression of Cdk4, facilitating the nuclear import of Cdk4, and sequestering Cdk4 in the nuclei as it simultaneously downregulates expression of p27 and facilitates the proteolysis of the molecule by phosphorylation.

Actins↗
PubMed1997

A distinct and parallel pathway for the nuclear import of an mRNA-binding protein.

Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. Here we have characterized a new pathway to the nucleus, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p bound peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways.

Amino Acid Sequence↗
PubMed2001

Phosphorylation-dependent nucleocytoplasmic shuttling of pancreatic duodenal homeobox-1.

Pancreatic duodenal homeobox-1 (PDX-1) is a homeodomain protein that plays an important role in the development of the pancreas and in maintaining the identity and function of the islets of Langerhans. It also regulates the expression of the insulin gene in response to changes in glucose and insulin concentrations. Glucose and insulin regulate PDX-1 by way of a signaling pathway involving phosphatidylinositol 3-kinase (PI 3-kinase) and SAPK2/p38. Activation of this pathway leads to phosphorylation of PDX-1 and its movement into the nucleus. To investigate the intracellular trafficking of PDX-1, immunocytochemistry was used to localize PDX-1 in the human beta-cell line NesPDX-1, in which PDX-1 is overexpressed, and in MIN6 beta-cells. In low-glucose conditions, PDX-1 localized predominantly to the nuclear periphery, with some staining in the cytoplasm. After stimulation with glucose, PDX-1 was present in the nucleoplasm. The translocation of PDX-1 to the nucleoplasm was complete within 15 min and occurred in 5-10 mmol/l glucose. Insulin and sodium arsenite, an activator of the stress-activated pathway, also stimulated PDX-1 movement from the nuclear periphery to the nucleoplasm. When cells were transferred between high glucose- and low glucose-containing medium, PDX-1 rapidly shuttled between the nuclear periphery and the nucleoplasm. Glucose- and insulin-stimulated translocation of PDX-1 to the nucleoplasm was inhibited by wortmannin and SB 203580, indicating that a pathway involving PI 3-kinase and SAPK2/p38 was involved; translocation was unaffected by PD 098959 and rapamycin, suggesting that neither mitogen-activated protein kinase nor p70(s6k) were involved. Arsenite-stimulated import of PDX-1 into the nucleus was inhibited by SB 203580 but not by wortmannin. Export from the nucleoplasm to the nuclear periphery was inhibited by calyculin A and okadaic acid, suggesting that dephosphorylation of PDX-1 was involved. These results demonstrated that PDX-1 shuttles between the nuclear periphery and nucleoplasm in response to changes in glucose and insulin concentrations and that these events are dependent on PI 3-kinase, SAPK2/p38, and a nuclear phosphatase(s).

Androstadienes↗
PubMed2001

Lipophilic statins can be osteogenic by promoting osteoblastic calcification in a Cbfa1- and BMP-2-independent manner.

Mevastatin (3-10 microM) and fluvastatin (0.1-10 microM), but not pravastatin, were found to promote calcification of MC3T3-E1 cells and their subclone MC4, in either the presence or absence of 3 mM inorganic phosphate stimulus. The mechanism of action was examined. Gel retardation assay and immunocytochemical analysis of core binding factor (Cbfa1) revealed that mevastatin and fluvastatin completed the nuclear export of Cbfa1, possibly thereby reducing the induction of the stably transfected p6OSE2-luc gene, and then promoted Cbfa1-independent calcification, which invariably occurred in both wild type and dominant negative Cbfa1-expressing cells. The induction of the bone morphogenetic protein-2 (BMP-2) gene promoter failed to respond to the statins. All the effects of the cell-permeable statins were negated by mevalonate pathway metabolites (geranylgeranylpyrophosphate > farnesylpyrophosphate > mevalonate) and reproduced by toxin B (a Rho-specific inhibitor), but not totally by Y27632 (a ROCK-specific inhibitor). The results suggest that lipophilic statins can be osteogenic by promoting Cbfa1- and BMP-2-independent calcification processes.

Animals↗