Induction of vascular endothelial growth factor expression and hypoxia-inducible factor 1alpha protein by the oxidative stressor arsenite

Recent evidence suggests that vascular endothelial growth factor (VEGF) expression is up-regulated by oxidative stressors through activation of hypoxia-inducible Factor 1 (HIF-1). To investigate whether this is a general phenomenon, we studied the effects of the sulfhydryl reagent arsenite on VEGF expression in human ovarian cancer cells. Arsenite potently induces the production of reactive oxygen species (ROS) in several cell systems and directly interacts with sulfhydryl groups of cellular thiols. We report that arsenite induces VEGF mRNA and protein levels in normoxic H134 and OVCAR-3 cells. Arsenite also increases HIF-1alpha protein levels, suggesting a role for HIF-1 in the induction of VEGF expression. Pretreatment with the ROS inhibitors catalase and mannitol attenuated arsenite-induced ROS production, but did not affect induction of VEGF mRNA and HIF-1alpha protein. In contrast, pretreatment with the thiol antioxidants glutathione or N-acetylcysteine completely abrogated both effects, whereas a potentiation was observed by depletion of intracellular glutathione. These results demonstrate that arsenite-induced VEGF mRNA and HIF-1alpha protein expression is independent of increased ROS production but critically regulated by the cellular reduced glutathione content. In addition, these data suggest the involvement of a thiol-sensitive mechanism in the regulation of VEGF mRNA expression and HIF-1alpha protein in human ovarian cancer cells.     

Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes

We have previously shown that influenza haemagglutinin (HA) acquires Endo H resistance en route to the cell surface after microinjection of its mRNA into Xenopus oocytes (Ceriotti, A. and A. Colman. 1989. J. Cell Biol. 109:1439-1444.) In this paper we use the injection of varying amounts of mRNA (0.05-5 ng/oocyte) to effect a 30-fold change in HA protein synthesis within the oocyte. Using the Endo H assay as an indicator of protein movement from the ER to the medial Golgi we find that this movement is reduced, sometimes dramatically, when intracellular HA levels fall. This reduction in movement is closely correlated with a decreased rate of trimer formation as assessed both by trypsin resistance and sedimentation analysis, leading us to conclude that trimer formation is not only, as has been shown before essential for ER-Golgi complex movement, but is the major rate limiting step in this movement. Interestingly at least 50% of unassembled HA monomers that accumulate after low HA synthesis can be rescued into trimers over 24 h later, after a second injection of concentrated HA mRNA. In contrast when we repeated this experiment with another membrane protein, the human low density lipoprotein, or with murine secretory immunoglobulin we found that the rate of movement was insensitive to the protein concentration. This latter result seemed surprising since earlier work had shown that unassembled IgG heavy chains (like monomeric HA) remain in the oocyte ER; however in these present experiments we have been unable to detect any unassembled heavy chains even at the lowest expression levels, indicating that tetramerization of Ig is much faster than trimerization of HA.     

Hepatocyte growth Factor/Scatter factor (HGF/SF) is a regulator of fibronectin splicing in MDCK cells: comparison between the effects of HGF/SF and TGF-beta1 on fibronectin splicing at the EDA region.

EDA-containing fibronectin (EDA + FN) is selectively produced under several physiological and pathological conditions requiring tissue remodeling, where cells actively proliferate and migrate. Only a few growth factors, such as transforming growth factor (TGF)-beta1, have been reported to regulate FN splicing at the EDA region. In the present study, we showed for the first time that hepatocyte growth factor/scatter factor (HGF/SF), which is mainly produced by mesenchymal cells and functions as a motogenic and mitogenic factor for epithelial cells, modulates FN splicing at the EDA region in MDCK epithelial cells. HGF/SF treatment increased the ratio of EDA + FN mRNA to mRNA of FN that lacks EDA (EDA - FN) (EDA+/EDA- ratio) more than TGF-beta1 treatment did: at a range from 0.02 to 20 ng/ml, HGF/SF increased the ratio in a dose-dependent manner by up to 2. 1-fold compared with nontreated control, while TGF-beta1 stimulated the EDA+/EDA- ratio by 1.5-fold at the optimum dose of 10 ng/ml. However, TGF-beta1 increased total FN mRNA levels by 3-fold at 10 ng/ml, but HGF/SF did not. We previously demonstrated that fibroblasts cultured at low cell density expressed more EDA + FN than those at high cell density. The same effect of cell density was also observed in MDCK cells. Furthermore, at low cell density, HGF/SF stimulated EDA inclusion into FN mRNA more effectively than did TGF-beta1, whereas at high cell density, TGF-beta1 was more potent than HGF/SF. Simultaneous treatment of cells with HGF/SF and TGF-beta1 synergistically stimulated EDA inclusion into FN mRNA. This stimulation of EDA inclusion into FN mRNA by HGF/SF led to increased EDA + FN protein production and secretion by cells, which was demonstrated by immunoblotting. Thus, our studies have shown that HGF/SF is an enhancer of EDA inclusion into FN mRNA as is TGF-beta1. However, these two factors were different in their effects at low and high cell densities and also in their effects on total FN mRNA levels.     

Cell-to-cell signaling in the regulation of procollagen expression in primary avian tendon cells

Summary High cell density is required for high procollagen expression (50% of total protein synthesis) in primary avian tendon (PAT) cells but the signaling mechanism that triggers this response has been difficult to decipher. By using a quantitative in situ hybridization assay for procollagen mRNA, cell density dependent changes in procollagen expression can be followed at the single cell level. PAT cells can then be shown to respond to the presence of their neighbors over ∼1-mm distance. The cell density signal remains effective independent of the medium volume to cell ratio but becomes sensitive to dispersion and dilution when the medium is agitated. PAT cells respond to a reduction in cell density, when neighboring cells are scraped away, by outgrwoth (∼1 mm) and reestablishment of a cell density gradient in cellular procollagen mRNA levels. However, removing neighboring cells while preventing migration off of their own extracellular matrix retards the drop in procollagen mRNA levels. The evidence, taken as a whole, is consistent with a model whereby the cell density signal is a loosely bound component of the cell layer thereby restricting its diffusion to two dimensions but making it susceptible to dispersion by medium agitation. This work was supported in part by grant CA 37958 from the National Institutes of Health, Bethesda, MD, and in part by the Office of Health and Environmental Research, U.S. Dept. of Energy, Washington, DC, under contract DE-AC03-76SF00098.     

Transport of beta-globin mRNA from nuclei of murine Friend erythroleukemia cells. Reversible inhibition of transport by the oxidizing sulfhydryl reagent o-iodosobenzoate

An in vitro assay system for analysis of beta-globin mRNA transport is described. Nuclei isolated from murine Friend erythroleukemia cells induced to synthesize globin mRNA, were incubated in micro-assays. By electrophoresis and hybridization analysis, released 9-S beta-globin mRNA was shown to be undegraded. After direct blotting, the released mRNA was quantified by hybridization with a labeled plasmid containing a beta-globin DNA restriction fragment. The inducibility of beta-globin mRNA transport corresponded to that previously reported for the release of rapidly labeled RNA in other assay systems. In contrast to the ineffectiveness of high concentrations of the sulfhydryl reagent iodoacetate, low concentrations of the oxidizing sulfhydryl reagent, o-iodosobenzoate, inhibited the release of beta-globin mRNA from nuclei of erythroleukemia cells, as well as the release of rapidly labeled RNA from rat liver nuclei. The inhibitory effect of the oxidizing agent on beta-globin mRNA transport could be reversed by postincubation of the nuclei with the reducing agent, dithiothreitol. The potential role of disulfide bond formation on RNA transport is discussed.     

Essential Sulfhydryl Group in the Transport-catalyzing Protein of the Hexose-Proton Cotransport System of Chlorella

The polyene antibiotic nystatin transforms the sugar-proton contransport system of Chlorella to a mere facilitated diffusion system. This experimental condition was used to test the sugar-translocating unit of the active uptake system for possible essential sulfhydryl groups. It could be shown that the catalyzed translocation of sugar is sensitive to the sulfhydryl-reactive compound N-ethylmaleimide. Sugar flow by passive leak as induced by the detergent Triton X-100 is not affected by sulfhydryl reagents. These results show that the sugar-translocating carrier protein possesses a sulfhydryl group, which is essential for its function.     

Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells.

A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant clone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that we previously described (Biochem. Biophys. Res. Commun. 121, 421-427, 1984) as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.     

Differential regulation of three glucose transporter genes in a renal epithelial cell line

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.     

Progesterone neuroprotection in spinal cord trauma involves up-regulation of brain-derived neurotrophic factor in motoneurons

Progesterone (PROG) provides neuroprotection to the injured central and peripheral nervous system. These effects may be due to regulation of myelin synthesis in glial cells and also to direct actions on neuronal function. Both types of cells express classical intracellular PROG receptors (PR), while neurons additionally express the PROG membrane-binding site called 25-Dx. In motoneurons from rats with spinal cord injury (SCI), PROG restores to normal the deficient levels of choline acetyl-transferase and of alpha3 subunit Na,K-ATPase mRNA, while levels of the growth associated protein GAP-43 mRNA are further stimulated. Recent studies suggest that neurotrophins are possible mediators of hormone action, and in agreement with this assumption, PROG treatment of rats with SCI increases the expression of brain-derived neurotrophic factor (BDNF) at both the mRNA and protein levels in ventral horn motoneurons. In situ hybridization (ISH) has shown that SCI reduces BDNF mRNA levels by 50% in spinal motoneurons, while PROG administration to injured rats (4mg/kg/day during 3 days, s.c.) elicits a three-fold increase in grain density. In addition to enhancement of mRNA levels, PROG increases BDNF immunoreactivity in perikaryon and cell processes of motoneurons of the lesioned spinal cord, and also prevents the lesion-induced chromatolytic degeneration of spinal cord motoneurons as determined by Nissl staining. Our findings strongly indicate that motoneurons of the spinal cord are targets of PROG, as confirmed by the expression of PR and the regulation of molecular parameters. PROG enhancement of endogenous neuronal BDNF could provide a trophic environment within the lesioned spinal cord and might be part of the PROG activated-pathways to provide neuroprotection. Thus, PROG treatment constitutes a new approach to sustain neuronal function after injury.     

Hormonal regulation of estrogen receptor messenger ribonucleic acid in T47Dco and MCF-7 breast cancer cells

Using a combination of hormone-binding assays, immunologic techniques, and mRNA hybridizations we have measured the estrogen receptor (ER) content and studied the hormonal regulation of ER mRNA in one estrogen responsive and one estrogen unresponsive breast cancer cell line, MCF-7 and T47Dco, respectively. Estradiol binding could be detected in cytosol from MCF-7 cells but not in T47Dco cells. However, when measured by an enzyme-linked immunosorbent assay, T47Dco cells were found to contain approximately 15 fmol ER/mg cytosolic protein or 10% of the ER content in MCF-7 cells. Immunologically reactive ER in T47Dco cells was indistinguishable in size (approximately equal to 68 KD) from the ER in MCF-7 cells, as shown by Western blotting using a monoclonal antihuman ER antibody. Quantification of ER mRNA in MCF-7 and T47Dco cells indicated that T47Dco cells contained approximately 50% of the ER mRNA levels found in MCF-7 cells. This basal level of ER mRNA in T47Dco cells was not decreased by estradiol treatment, as opposed to in MCF-7 cells where estradiol caused 40-60% decrease in the ER mRNA expression. Also, estradiol did not increase the progesterone receptor (PR) mRNA levels in T47Dco cells whereas in MCF-7 cells an approximately 5-fold increase of the PR mRNA levels occurred after estradiol treatment. However, incubation of the cells with the synthetic progestin R5020 decreased the ER mRNA levels to approximately the same degree in both cell lines. In conclusion, we have shown that estrogen down-regulates ER mRNA and up-regulates PR mRNA in MCF-7 cells. Neither of these estrogenic effects were seen in T47Dco cells. It appears that the steroid-resistance in T47Dco cells does not occur as a consequence of a complete absence of ER mRNA or protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.     

Cellular density regulation of plasminogen gene expression in mouse hepatocytes

The liver produces a variety of proteins including plasminogen. Plasminogen is pro-enzyme that is converted into plasmin by plasminogen activator. Plasmin has a broad substrate spectrum and participates in several biological processes, such as fibrinolysis, tissue remodeling, cell migration, angiogenesis and embryogenesis. In the present study, the regulation of plasminogen expression in mouse hepatocytes was investigated in the primary culture system. Expression level of plasminogen mRNA in the culture at the low cell density condition (0.2 x 10(5) cells / cm(2)) was compared with that at the high cell density condition (1.0 x 10 (5) cells / cm(2)). In the low cell density culture, the expression level of plasminogen mRNA decreased by a time-dependent manner. However, mRNAs for albumin and alpha(2)-antiplasmin were not influenced by the low cell density culture. On the other hand, in the high cell density culture, plasminongen mRNA expressed constantly as well as albumin and alpha(2)-antiplasmin mRNAs. Thus, the decrease in plasminogen mRNA expression could specifically occur when the density of hepatocytes was low. The down-regulation of plasminogen mRNA in the low cell density culture is not observed in the presence of cycloheximide, suggesting that the de novo protein synthesis is required for the regulatory mechanism. These findings indicate that the expression of plasminogen mRNA from hepatocyte is dependent on the cell density and the stimulation by cell-cell contact may be an important factor for the constitutive expression of plasminogen gene in hepatocytes.