Transcription-dependent Mechanism Research Articles

Neuronal activity-dependent cell survival mediated by transcription factor MEF2.

During mammalian development, electrical activity promotes the calcium-dependent survival of neurons that have made appropriate synaptic connections. However, the mechanisms by which calcium mediates neuronal survival during development are not well characterized. A transcription-dependent mechanism was identified by which calcium influx into neurons promoted cell survival. The transcription factor MEF2 was selectively expressed in newly generated postmitotic neurons and was required for the survival of these neurons. Calcium influx into cerebellar granule neurons led to activation of p38 mitogen-activated protein kinase-dependent phosphorylation and activation of MEF2. Once activated, MEF2 regulated neuronal survival by stimulating MEF2-dependent gene transcription. These findings demonstrate that MEF2 is a calcium-regulated transcription factor and define a function for MEF2 during nervous system development that is distinct from previously well-characterized functions of MEF2 during muscle differentiation.

Expression of the T Cell Antigen Receptor ζ Chain following Activation Is Controlled at Distinct Checkpoints: IMPLICATIONS FOR CELL SURFACE RECEPTOR DOWN-MODULATION AND RE-EXPRESSION

The multisubunit T cell antigen receptor (TCR) is involved in antigen recognition and signal transduction, leading to T cell activation and rapid down-modulation of the cell surface expressed TCRs. Although the levels of TCR cell surface expression are pivotal to the efficiency and duration of the immune response, the molecular mechanisms controlling TCR down-modulation and re-expression upon activation, remain obscure. Here, we provide a biochemical characterization of the regulatory mechanisms governing TCR expression following long-term T cell activation. We focused primarily on the TCR zeta chain, as this is considered the limiting factor in TCR complex formation and transport to the cell surface. We found that following TCR-mediated activation zeta mRNA is up-regulated by a transcription-dependent mechanism. Concomitantly, zeta protein levels are modified according to a biphasic pattern: rapid degradation coinciding with TCR cell surface down-regulation, followed by a rebound to normal levels 24 h subsequent to T cell activation. Even though there are adequate levels of all the TCR subunits within the cell following 24 h of activation, TCR cell surface expression remained very low, provided the activating antibody is continuously present. Correlative with the latter, we detected a previously undescribed monomeric form of the zeta chain. This form could be indicative of adverse endoplasmic reticulum conditions affecting correct protein folding, dimerization, and TCR assembly, all critical for optimal receptor surface re-expression. Cumulatively, our results indicate that the levels of TCR expression following activation, are tightly controlled at several checkpoints.

Sodium butyrate inhibits the phosphorylation of the retinoblastoma gene product in mouse fibroblasts by a transcription-dependent mechanism

In the chemically transformed mouse fibroblasts BP-A31, the retinoblastoma protein (pRB) is hypophosphorylated at quiescence and becomes hyperphosphorylated after approximately 6 h of serum stimulation. Phosphorylation of pRb was blocked if sodium butyrate was added together with serum or within 3 h afterwards. Actinomycin D added 3 h after serum stimulation did not prevent pRb phosphorylation, but it reversed the inhibitory effect of butyrate. These observations indicate that sodium butyrate acts by turning on the expression of gene(s) coding for proteins which prevent the accumulation of hyperphosphorylated pRb. Such butyrate-induced inhibitor(s) may interfere with the phosphorylation of pRb by cyclin-dependent kinases. Treatment of quiescent BP-A31 cells with serum in the presence of sodium butyrate has led to an increased cell content of the Waf1/CIP1 mRNA (coding for a cyclin-dependent) kinase inhibitory protein) compared with serum alone, suggesting a possible role of p21Waf1/CIP1. In contrast, the mitogen activated protein kinase (enzyme which has been shown to phosphorylate pRb) was constitutively active in BP-A31 cells, and its activity was not significantly affected by a < or = 3h incubation with sodium butyrate.

Adipocyte differentiation selectively represses the serum inducibility of c-jun and junB by reversible transcription-dependent mechanisms.

Nonterminally differentiated 3T3 T adipocytes are resistant to growth stimulation by 10% (vol/vol) fetal bovine serum even though they can be induced to proliferate with extremely high serum concentrations. We now report that in adipocytes 10% fetal bovine serum also fails to typically induce c-jun or junB. Rather, after 10% fetal bovine serum treatment, c-jun and junB expression is markedly repressed after a brief initial slight induction. Gel mobility shift studies confirm that AP-1 DNA binding activity is inhibited in adipocytes. Repression in c-jun and junB inducibility in adipocytes results from transcriptional mechanisms, can be reversed by treatment with protein synthesis inhibitors or higher serum concentrations, and does not affect c-fos or c-myc expression. These data suggest that adipocyte differentiation selectively and transcriptionally represses the inducibility of c-jun and junB so as to decrease the cell's ability to proliferate in response to 10% fetal bovine serum.

The type II "receptor" as a decoy target for interleukin 1 in polymorphonuclear leukocytes: characterization of induction by dexamethasone and ligand binding properties of the released decoy receptor.

Whereas the signaling function of the interleukin 1 (IL-1) receptor type I (IL-1R I) has been well documented, the type II "receptor" has been suggested to act as a decoy target for this cytokine. Since IL-1 may represent a key target of the immunomodulatory and antiinflammatory properties of glucocorticoids (GC), the aim of this study was to investigate the effects of dexamethasone (Dex) on IL-1R expression in human polymorphonuclear leukocytes (PMN), which express predominantly the type II molecule (IL-1R II). We found that Dex augments the levels of steady state transcripts encoding the IL-1R I and, most prominently, those of IL-1R II. Dex induced both transcripts via transcription-dependent mechanisms and by prolongation of the mRNAs half-lives. Inhibition of protein synthesis superinduced basal and Dex-augmented IL-1R II mRNA, whereas it completely inhibited the induction by Dex of IL-1R I transcripts. Induction of IL-1R II mRNA by Dex was associated with augmented membrane expression and release of the type II IL-1 binding molecule. This effect was mediated by the GC receptor. Other steroids (17 beta-estradiol, progesterone, and testosterone) were ineffective. The concentrations of IL-1 alpha and IL-1 receptor antagonist required to displace the binding of IL-1 beta to the soluble form of the decoy molecule induced by Dex from PMN were, respectively, 100 and 2 times higher compared with IL-1 beta. The induction by Dex of the type II receptor, a decoy molecule for IL-1, may contribute to the immunosuppressive and antiinflammatory activities of Dex.

Transcriptional activation of ornithine decarboxylase in adult and neonatal hippocampal slices

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine synthesis and is regulated by both transcription-dependent and transcription-independent mechanisms. We compared the effects of asparagine, an amino acid previously shown to increase ODC activity in adult hippocampal slices, on ODC mRNA and activity in adult and neonatal hippocampal slices. In addition, we evaluated the effects of asparagine on ODC activity following seizure activity elicited by systemic administration of kainic acid (KA) in both adult and neonatal rats. Asparagine produced an increase in ODC gene expression and activity in both adult and neonatal hippocampal slices. The increase in ODC activity elicited by asparagine in hippocampal slices was the same in control animals as in animals sacrificed 16 h after KA-induced seizure activity. The asparagine-elicited increase in ODC activity in neonatal and adult hippocampal slices was blocked by the RNA synthesis inhibitor, actinomycin D. Finally, polyamines produced an inhibition of ODC activity in neonatal hippocampal slices. The results indicate that the regulation of the expression and activity of ODC is similar in neonatal and adult hippocampus.

NGF induces the expression of the VGF gene through a cAMP response element

NGF is a peptide growth factor that plays a key role in the differentiation and survival of neurons in both the PNS and CNS. NGF acts through both transcription-dependent and transcription-independent mechanisms to regulate the differentiation of PC12 cells. To better understand the regulation of gene expression by NGF, we have defined a cis-acting sequence that is immediately upstream of the transcription start site of the VGF (a2/NGF33.1) gene that is required for induction by NGF. Within this sequence is a consensus cAMP response element (CRE) embedded in a 14 base pair palindrome. Mutations in this CRE eliminate induction of the VGF gene both by NGF and by agents that act via cAMP. Although this sequence confers transcriptional induction by both NGF and cAMP, it is not sufficient to allow induction by epidermal growth factor, acidic or basic fibroblast growth factor, or phorbol 12-myristate 13-acetate (PMA). Thus, this sequence defines an element that is selectively activated by NGF and cAMP. Promoter fragments from the VGF gene that include the core CRE efficiently bind the inducible transcription factor CREB, while fragments bearing mutations that eliminate NGF and cAMP inducibility fail to do so. Sequence comparisons and hybridization studies indicate that there are at least two alternatively spliced forms of VGF mRNA, and the accumulation of both of these forms is similarly regulated by NGF and cAMP.

Hormone specific regulation of natural killer cells by cortisol Direct inactivation of the cytotoxic function of cloned human NK cells without an effect on cellular proliferation

Corticosteroids have previously been reported to partially inhibit the natural cytotoxic activity of peripheral blood lymphocytes. However, since only a few percent of peripheral lymphocytes are natural killer (NK) cells, it has not been possible to determine whether corticosteroids directly inhibit NK cells or mediate this effect via other cell types. This report documents direct functional inactivation, but unimpeded proliferation, of cloned human NK cells by subphysiologic levels of cortisol. In contrast, high concentrations of testosterone, progesterone or estradiol had no significant effect on proliferation or cytotoxic activity of the cloned NK cells. The kinetics of inhibition of NK function by cortisol are consistent with a transcription-dependent mechanism.

Cell surface Thy-1-cross-reactive glycoprotein in cultured PC12 cells: modulation by nerve growth factor and association with the cytoskeleton

In cultured PC12 rat pheochromocytoma cells, nerve growth factor (NGF) selectively stimulated the incorporation of [3H]fucose into a glycoprotein of apparent Mr approximately 25,000, as determined by sodium dodecyl sulfate-gel electrophoresis. Neither dibutyryl cyclic adenosine 3':5'-monophosphate nor epidermal growth factor mimicked this effect. Using gradient gels, the affected protein was resolved into two closely migrating bands. Enhancement of labeling was present by 1 to 2 days of treatment with NGF and was maximal after 4 to 7 days. Short-term extraction of PC12 cell monolayer cultures with Triton X-100 left the 25,000-dalton glycoprotein associated with the detergent-resistant cytoskeletal fraction. The Mr approximately 25,000 glycoprotein was shown to be immunologically cross-reactive with Thy-1.1 antigen by indirect immunoprecipitation with monoclonal Thy-1.1 antibodies. Anti-Thy-1.1 labeling, as demonstrated by indirect immunofluorescence, was distributed on PC12 cell bodies and along neuritic processes and remained attached to the cytoskeleton as part of the surface lamina of cells treated and untreated with NGF. The selective increase of the Mr approximately equal to 25,000 glycoprotein in NGF-treated cultures was also paralleled by increases in material immunoprecipitated from fucose-labeled cells with anti Thy-1.1 monoclonal antibodies. Immunoprecipitations with extracts of [35S]methionine-labeled cultures indicated that NGF causes an increase in synthesis, rather than merely glycosylation, of the 25,000-dalton/Thy-1-cross-reactive protein. The effect of NGF on this protein was blocked by inhibitors of RNA synthesis, suggesting involvement of a transcription-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)