The cytoskeletal network controls c-Jun translation in a UTR-dependent manner.

The cytoskeleton is a dynamic network that undergoes restructuring during various cellular events, influencing cell proliferation, differentiation, and apoptosis. Here, we report that accumulation of c-Jun, a member of the AP1 family of transcription factors that play a key role in normal and aberrant cell growth, dramatically increases upon depolymerization of the cytoskeleton, and that, unexpectedly, this increase is controlled translationally. Depolymerization of the actin or microtubule network induces an increase in c-Jun accumulation with no corresponding increase in c-Jun mRNA or in the half-life of the c-Jun protein, but rather in the translatability of its transcript. This increase is mediated by the untranslated regions (UTRs) of c-Jun mRNA, and is not dependent on activated mitogen-activated protein kinase pathways. This novel mechanism of c-Jun regulation might be relevant to physiological conditions in which c-Jun plays a pivotal role.

Basic fibroblast growth factor: a potential inhibitor of glutamine synthetase expression in injured neural tissue.

Basic fibroblast growth factor (bFGF) was recently shown to promote the survival of neural cells and tissues, raising hopes for its therapeutic potential in degenerative disorders of the CNS. Here we examine the effect of bFGF on the expression of glutamine synthetase, a key enzyme in the detoxification of the neurotransmitter glutamate. Expression of this enzyme is regulated by systemic glucocorticoids and, in chick neural retina tissue, is restricted to Müller glial cells. We report that exogenous supply of bFGF to retinal explants inhibits hormonal induction of glutamine synthetase expression. This inhibition appears to be mediated by the c-Jun protein which accumulated, in response to bFGF, exclusively in Müller glial cells. Ischemic conditions, which reportedly stimulate the release of endogenous bFGF, also led to an increase in c-Jun protein and a decline in glutamine synthetase expression. This decline could be competitively prevented by a soluble fibroblast growth factor receptor but not by a soluble epidermal growth factor receptor. The finding that endogenous release of bFGF or its exogenous supply down-regulates glutamine synthetase expression suggests that in addition to its reported neuroprotective effect, bFGF may exacerbate glutamate mediated neurotoxicity through direct down-regulation of glutamine synthetase.

Doxorubicin and a butyric acid derivative effectively reduce levels of BCL-2 protein in the cells of chronic lymphocytic leukemia patient.

B-chronic lymphocytic leukemia (B-CLL) is a disease caused primarily by defects in the apoptosis mechanism. AN-9, a butyric acid (BA) derivative, is a potent differentiating and an anti-cancer drug that induces apoptosis in HL-60 cells. Herein we show the affect of AN-9, alone and in combination with doxorubicin, on cell cultures from B-CLL patients. Cells from 17 patients were cultured and tested for viability, apoptosis, bcl-2 and bax protein expression. Exposure of B-CLL cell cultures to AN-9 was accompanied by apoptosis and a marked viability loss (up to 46%, p=0.0017). AN-9 reduced up to 51% (p=0.0017) the levels of bcl-2 in 57% of the cultures that express bcl-2. The combination of low concentrations of AN-9 and doxorubicin more than additively enhanced apoptosis and reduced bcl-2 levels in B-CLL cultures which were resistant to AN-9. AN-9 enhanced bax expression up to 58%(p=0.008) in cultures from 53% of the patients, but had no effect on bax levels when combined with doxorubicin. In conclusion, AN-9 alone reduced bcl-2 and enhanced bax expression in cultures from B-CLL patients, and the reduction of bcl-2 levels in combination with doxorubicin was greater than additive. These results may be beneficial in possible future combination therapy with AN-9 in B-CLL.

Glucocorticoid control of glial gene expression.

The glucocorticoid signaling pathway is responsive to a considerable number of internal and external signals and can therefore establish diverse patterns of gene expression. A glial-specific pattern, for example, is shown by the glucocorticoid-inducible gene glutamine synthetase. The enzyme is expressed at a particularly high level in glial cells, where it catalyzes the recycling of the neurotransmitter glutamate, and at a low level in most other cells, for housekeeping duties. Glial specificity of glutamine synthetase induction is achieved by the use of positive and negative regulatory elements, a glucocorticoid response element and a neural restrictive silencer element. Though not glial specific by themselves, these elements may establish a glial-specific pattern of expression through their mutual activity and their combined effect. The inductive activity of glucocorticoids is markedly repressed by the c-Jun protein, which is expressed at relatively high levels in proliferating glial cells. The signaling pathway of c-Jun is activated by the disruption of glia-neuron cell contacts, by transformation with v-src, and in proliferating retinal cells of early embryonic ages. The c-Jun protein inhibits the transcriptional activity of the glucocorticoid receptor and thus represses glutamine synthetase expression. This repressive mechanism might also affect the ability of glial cells to cope with glutamate neurotoxicity in injured tissues.

A silencer element in the regulatory region of glutamine synthetase controls cell type-specific repression of gene induction by glucocorticoids.

Glutamine synthetase is a key enzyme in the recycling of the neurotransmitter glutamate. Expression of this enzyme is regulated by glucocorticoids, which induce a high level of glutamine synthetase in neural but not in various non-neural tissues. This is despite the fact that non-neural cells express functional glucocorticoid receptor molecules capable of inducing other target genes. Sequencing and functional analysis of the upstream region of the glutamine synthetase gene identified, 5' to the glucocorticoid response element (GRE), a 21-base pair glutamine synthetase silencer element (GSSE), which showed considerable homology with the neural restrictive silencer element NRSE. The GSSE was able to markedly repress the induction of gene transcription by glucocorticoids in non-neural cells and in embryonic neural retina. The repressive activity of the GSSE could be conferred on a heterologous GRE promoter and was orientation- and position-independent with respect to the transcriptional start site, but appeared to depend on a location proximal to the GRE. Gel-shift assays revealed that non-neural cells and cells of early embryonic retina contain a high level of GSSE binding activity and that this level declines progressively with age. Our results suggest that the GSSE might be involved in the restriction of glutamine synthetase induction by glucocorticoids to differentiated neural tissues.

The cytoskeletal network controls c-Jun expression and glucocorticoid receptor transcriptional activity in an antagonistic and cell-type-specific manner.

The physical and functional link between adhesion molecules and the cytoskeletal network suggests that the cytoskeleton might mediate the transduction of cell-to-cell contact signals, which often regulate growth and differentiation in an antagonistic manner. Depolymerization of the cytoskeleton in confluent cell cultures is reportedly sufficient to initiate DNA synthesis. Here we show that depolymerization of the cytoskeleton is also sufficient to repress differentiation-specific gene expression. Glutamine synthetase is a glia-specific differentiation marker gene whose expression in the retinal tissue is regulated by glucocorticoids and is ultimately dependent on glia-neuron cell contacts. Depolymerization of the actin or microtubule network in cells of the intact retina mimics the effects of cell separation, repressing glutamine synthetase induction by a mechanism that involves induction of c-Jun and inhibition of glucocorticoid receptor transcriptional activity. Depolymerization of the cytoskeleton activates JNK and p38 mitogen-activated protein kinase and induces c-Jun expression by a signaling pathway that depends on tyrosine kinase activity. Induction of c-Jun expression is restricted to Müller glial cells, the only cells in the tissue that express glutamine synthetase and maintain the ability to proliferate upon cell separation. Our results suggest that the cytoskeletal network might play a part in the transduction of cell contact signals to the nucleus.

Effect of the cytostatic butyric acid pro-drug, pivaloyloxymethyl butyrate, on the tumorigenicity of cancer cells.

Previously we have shown that pivaloyloxymethyl butyrate (AN-9), a pro-drug of butyric acid (BA), is a differentiation-inducing agent in a variety of cells. In this report, we demonstrate that AN-9 is a cytostatic but not cytotoxic agent in a myelomonocytic cell line (WEHI); thus, the cells were growth-arrested and differentiated. These late changes in the cells were preceded by changes in the expression of the early regulatory genes, c-myc and c-jun. Although initiation of all these events had already occurred after 1 h exposure to AN-9, the tumorigenicity of these cells tested in Balb/c mice was not affected. A marked reduction in the tumorigenicity of AN-9-treated cells was observed after 4 h of exposure. Exposure of the highly metastatic subclone of Lewis lung carcinoma (3LLD122) to AN-9 resulted in a very pronounced effect on the tumorigenicity of these cells tested in C57BL mice. Unlike WEHI cells, the tumorigenicity of 3LLD122 was almost completely diminished after 1 h of exposure. In both cell types a 10-fold higher concentration of BA did not affect the tumorigenicity of the cells as did AN-9.

Developmental changes in the expression and compartmentalization of the glucocorticoid receptor in embryonic retina.

Inducibility by glucocorticoids of the glutamine synthetase gene in chicken embryo retina and the transcriptional activity of the glucocorticoid receptor (GR) greatly increase between embryonic days 6 and 10 (E6, E10), although the level of GR does not markedly change during that time. This apparent discrepancy was investigated by examining the pattern of GR expression in undifferentiated E6 retina and in E10 retina, which consists mostly of differentiated cells. Two GR isoforms, 90 and 95 kDa, were found to be expressed at both of these ages at a similar total level but in different proportions: in E6 retina the level of the 90-kDa isoform was higher, whereas in E10 retina the 95-kDa receptor was higher. However, following treatment of the retinas with cortisol, the 95-kDa isoform became the predominant receptor at both ages. Immunohistochemical analysis revealed that the cellular localization of GR markedly changed in the course of development: in the undifferentiated E6 retina GR was expressed in virtually all cells, whereas in the more differentiated E10 and E12 retina, GR was detected only in Müller glia cells. The latter represent approximately 20% of the cells in this tissue and are the only cells in which glucocorticoid hormone induces the glutamine synthetase gene. We suggest that the compartmentalization of GR in Müller glia is a major aspect of the mechanism that modulates receptor activity during retina development and results in the temporal increase in the inducibility of glutamine synthetase and its specific localization in Müller glia cells.

Molecular basis for differential expression of glutamine synthetase in retina glia and neurons.

Glutamine synthetase (GS) is a differentiation marker of retina glial cell. It is expressed in the chicken neural retina at a particularly high level, is inducible by glucocorticoids and is always confined to Müller glia. This study investigated the molecular basis for tissue and cell-type specific expression of the GS gene. A high level of GS expression in the retina was found to coincide with the accumulation of a relatively high level of GS mRNA in this tissue. The gliatoxic agent alpha-aminoadipic acid, which can selectively destroy glia cells, was used to demonstrate that restriction of GS induction to Müller glia is controlled at a transcriptional level. Cortisol could induce accumulation of GS mRNA and transcription of the GS gene in Müller glia but not in retina neurons. Glia and neurons were also found to differ in their ability to express the glucocorticoid inducible CAT construct, p delta G46TCO, which is controlled by a 'simple GRE' promoter. When introduced into cells of retina tissue, this construct was cortisol-inducible in glia whereas in neurons it was only slightly inducible or not at all. Introduction of a glucocorticoid receptor expression vector into the cells facilitated induction of the CAT construct in neurons. Analysis by immunoblotting revealed that expression of the glucocorticoid receptor protein is predominantly restricted to Müller glia. These results suggest that differential levels of glucocorticoid receptor expression in glia and neurons might be the basis for cell-type specific induction of GS.

Molecular control of glutamine synthetase expression in the developing retina tissue.

Glutamine synthetase is a differentiation marker of the neural retina, whose expression is restricted to Müller glia cells, is inducible by glucocorticoids and is dependent on tissue development. The retina tissue acquires the competence to express GS in response to glucocorticoids with development, although the level of hormone binding activity in the cells does not alter with age. Using CAT constructs that are controlled by "simple GRE" promoters we demonstrated that glucocorticoid receptor transcription activity in retina cells increases with development. The increase in receptor activity correlates directly with the increase in inducibility of the glutamine synthetase gene and inversely with the rate of retina cell proliferation. At early developmental ages, when retina cells are still proliferating, the glucocorticoid receptor is transcriptionally inactive and glutamine synthetase expression cannot be induced. Receptor activity increases progressively with development and by day 12, when cell proliferation ceases, competence for glutamine synthetase induction is high. This competence for glutamine synthetase induction can be repressed by overexpressing the oncogene v-src, which stimulates retina cell proliferation. We discuss possible mechanisms for developmental-dependent modulation of glucocorticoid receptor transcriptional activity.

Foreign body granulomatous inflammation increases the sensitivity of splenocytes to immunomodulation by 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3, the active metabolite of vitamin D, partially inhibits antigen and mitogen-driven lymphocyte stimulation. We studied the effect of granulomatous inflammation on the sensitivity of lymphocytes to 1,25-dihydroxyvitamin D3 in vitro, measuring the inhibitory effect of 1,25-dihydroxyvitamin D3 on mitogenesis of splenocytes of mice with chronic inflammation induced by subcutaneous injection of talc. Systemic manifestations of the local inflammation included loss in body weight, splenomegaly, enhanced DNA synthesis by freshly isolated splenocytes and enhanced prostaglandin secretion by activated splenocytes. Splenocytes from animals with local inflammation were more susceptible to inhibition by 1,25-dihydroxyvitamin D3, but not by prostaglandin E2. This increased sensitivity to 1,25-dihydroxyvitamin D3 was abolished by blocking prostaglandin synthesis in splenocyte cultures with indomethacin and was restored by adding prostaglandin E2. This effect cannot be attributed to enhanced prostaglandin synthesis in the presence of 1,25-dihydroxyvitamin D3, but is probably due to a qualitative change in the response of splenocytes from inflamed animals to the combined action of 1,25-dihydroxyvitamin D3 and prostaglandin E2.

Developmental control of glucocorticoid receptor transcriptional activity in embryonic retina.

In chicken embryo retina, competence for induction of the glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2] gene by glucocorticoid hormones increases progressively with development; this competence is minimal in 6-day retina (E6) and high by day 10 (E10). Because the level of glucocorticoid receptors (GRs) in the retina does not increase during that time, we investigated whether the transcriptional activity of GR increased between days 6 and 10 of development. The glucocorticoid-inducible chloramphenicol acetyltransferase (CAT) constructs 2GRE-37TK and p delta G46TCO, which contain glucocorticoid-responsive elements attached to a TATA box and to the thymidine kinase promoter, respectively, were transfected into E6 and E10 retinas, and their inducibility was examined. CAT expression could be induced in the transfected E10 retina but was not induced in the transfected E6 retina. However, induction was obtained also in E6 retina after cotransfection with a GR expression vector. Noninducible CAT constructs (pRSV-CAT, pSV2CAT, and pBLCAT2) were expressed at both ages at similar levels. The CAT construct pGS2.1CAT, which is controlled by the upstream sequence of the chicken glutamine synthetase gene, could be induced in E10 retina but was not induced in E6 retina; however, cotransfection with the GR expression vector resulted in induction of pGS2.1CAT also in E6 retina. We interpret these results as showing that the transcriptional activity of GR in embryonic retina is developmentally controlled and suggest that its increase is causally implicated in the development of competence for glutamine synthetase induction.

Word identification in isolation and in context by college dyslexic students.

College dyslexic students (DYS) were compared to chronological age (CA)-matched and to reading age (RA)-matched control groups on tasks assessing naming of words and nonwords, regular and irregular words, and the use of context in word identification. The DYS group had the slowest naming latency for words in all tasks. In addition, they had extreme difficulty in naming nonwords, which in terms of the dual-route model for word recognition indicates impairment in the indirect route to the lexicon. However, they displayed a regularity effect in reading regular and irregular words and thus apparently utilized the indirect route in reading words. Correlational data supported the conclusion that the data are in conflict with the traditional dual-route model, and an alternative conceptualization is suggested. The use of context is discussed in terms of the interactive-compensatory model (Stanovich, 1980) and findings were generally supportive of the model. The DYS subjects of the present study appeared to be different from the RA controls and their performance did not support a developmental-lag model for explaining their reading problems.