Incomplete inactivation of voltage-dependent K+ channels in human B lymphoma cells.

The voltage-dependent K (KV) channel in Daudi human B lymphoma cells was characterized by using patch-clamp techniques. Whole-cell voltage-clamp experiments demonstrated that cell membrane depolarization induced a transient (time-dependent) outward current followed by a steady-state (time-independent) component. The time-dependent current resembled behavior of the type n channel, such as use dependence and a unique blockade by tetraethylammonium (TEA). Both time-dependent and time-independent currents were blocked by quinine with a similar IC50 (14.2 mM and 12.6 mM). Treatment with antisense oligonucleotide of human Kv1.3 gene significantly reduced both currents by 80%. Single-channel experiments showed that only one type of KV channel was recorded with a unitary conductance of approximately 19 pS. Consistent with whole-cell recordings, the channel activity in cell-attached patches remained in response to prolonged depolarization, and the remaining channel activity was blocked by quinine, but not TEA. Channel activity was scarcely seen in cell-attached patches after antisense treatment. Whole-cell current-clamp data showed that TEA, which blocks only the time-dependent current, caused a slight decrease in the membrane potential. In contrast, quinine and antisense, which block both time-dependent and -independent currents, strongly reduced the membrane potential. These data together suggest that the KV channel in Daudi cells does not completely inactivate and that the remaining channel activity due to this incomplete inactivation appears to be primarily responsible for maintaining the membrane potential.

Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts.

PURPOSE: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. METHODS AND MATERIALS: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1 and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. RESULTS: X-ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. CONCLUSIONS: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.

Inhibition of NFkB DNA binding activity by glucocorticoids in rat brain.

The influence of glucocorticoids on the transcription factor NFkB was investigated by using the gel mobility shift assay with nuclear extracts prepared from rat cerebral cortex and hippocampus after a variety of in vivo treatments. Following stimulation with each of three treatments, kainate, pilocarpine, or lithium plus pilocarpine-induced seizures, NFkB DNA binding activity was significantly greater in the cortex and hippocampus from adrenalectomized than from adrenal-intact rats. These results indicate that in rat brain glucocorticoids inhibit NFkB activity in addition to the previously reported inhibition of the transcription factor AP-1 (activator protein 1). Impairment of stimulus-induced transcription factor activity may contribute to the deleterious effects of prolonged elevations of glucocorticoids on neuronal function.

Diurnal variation in kainate-induced AP-1 activation in rat brain: influence of glucocorticoids.

The large diurnal rhythm of circulating glucocorticoid levels was used to determine if physiological fluctuations of glucocorticoids were capable of modulating kainate-induced immediate early gene (IEG) activation, measured as AP-1 DNA binding activity, in rat brain since administered dexamethasone previously had been shown to be inhibitory. AP-1 activity in the cerebral cortex 1.5 h after kainate treatment measured at 08.00 h (4.9-fold control) was more than twice the stimulation obtained at 16.00 h (1.8-fold). These times of day are associated with reported low and high levels of circulating glucocorticoids at 08.00 and 16.00 h, respectively. To test if there was a causal relationship, kainate-induced AP-1 activity was measured at both times in adrenalectomized rats. Adrenalectomy abolished the attenuation of the response to kainate found in intact rats at 16.00 h, indicating that the diurnal fluctuations in circulating glucocorticoids contribute to modulation of IEG responses to kainate. Neither AP-1 activity in the hippocampus nor cyclic AMP response element activation in either brain region measured after kainate treatment was influenced by the time of day or by adrenalectomy. Immunoprecipitation of glucocorticoid receptors from cortical nuclear extracts co-precipitated c-Jun, indicating that the mechanism accounting for the suppression of AP-1 activity by glucocorticoids may involve direct interactions between activated glucocorticoid receptors and AP-1 constituent proteins. These results extend previous reports that administered glucocorticoids inhibit AP-1 activity by demonstrating that this occurs with endogenous glucocorticoids as a consequence of the circadian rhythm of circulating glucocorticoids and demonstrate that responses to kainate vary dependent upon the time of day.

Dexamethasone attenuates kainate-induced AP-1 activation in rat brain.

The goal of this investigation was to determine if administration of the synthetic glucocorticoid dexamethasone modulates rat brain AP-1 DNA binding activity. Treatment with the selective excitatory amino acid agonist kainate was used to activate AP-1 formation. Kainate (12 mg/kg) administration induced a biphasic activation of AP-1 in rat cerebral cortex and hippocampus with maximal levels observed at 1.5 h and 4.5 h and lower levels at 3 h and 6 h. Kainate also induced biphasic increases in the concentrations of some of the AP-1 constituent proteins (immediate early gene protein products), with initial increases of c-Jun, Fos, and Jun B occurring at 1.5 h and secondary larger increases at 4.5 h, but the level of Jun D was not altered by kainate treatment. Pretreatment with dexamethasone (1 mg/kg) reduced AP-1 activity at both 1.5 h and 4.5 h after kainate administration in both brain regions. Dexamethasone pretreatment did not modify the concentrations of the AP-1 constituent proteins obtained after kainate administration except for a reduction of Jun B levels 1.5 h after kainate. These results demonstrate that elevated glucocorticoid levels reduce the stimulation by kainate of AP-1 activity in rat cortex and hippocampus without causing corresponding decreases in the levels of immediate early gene proteins. Binding of the activated glucocorticoid receptor to c-Jun or Fos is likely to contribute to the decreased AP-1 DNA binding activity following dexamethasone treatment.

A peptide encoding the c-Jun delta domain inhibits the activity of a c-jun amino-terminal protein kinase.

Evidence suggests that the c-Jun protooncogene delta (delta) domain (amino acids 31-60) helps regulate the transcriptional activating capacity of c-Jun by modulating the amino-terminal phosphorylation of this protein. By using a peptide encoding the delta domain and purified amino-terminal c-Jun protein kinase, we demonstrate that the delta domain peptide inhibits phosphorylation of the amino terminus of both c-Jun and the related protein JunD. The delta domain peptide inhibited the activation of the c-Jun amino-terminal protein kinase by phorbol esters in permeabilized U937 leukemic cells. Mutation of c-Jun followed by transfection into U937 leukemic cells demonstrated that partial deletions of the delta domain are sufficient to block phosphorylation of the amino terminus of c-Jun. In vitro deletion of the amino-terminal (amino acids 31-44) half of the delta domain inhibited the phosphorylation of c-Jun. However, deletion of the carboxyl-terminal (amino acids 45-60) half only partially inhibited c-Jun phosphorylation. Therefore, these results indicate that the delta domain sequence is an important regulator of c-Jun amino-terminal phosphorylation.

Multiple signal transduction pathways mediate c-Jun protein phosphorylation.

A variety of protein kinases, including pp42 and pp54 mitogen-activated protein (MAP) kinases, p34cdc2, and a partially purified protein kinase from 4 beta-phorbol 12-myristate 13 alpha-acetate (PMA)-treated U937 cells have been shown to phosphorylate the NH2-terminal activation domain of c-Jun in vitro. To investigate the role of pp42 MAP kinase in mediating c-Jun phosphorylation in vivo, we have treated U937 monocytic leukemia cells with a variety of pharmacological agents, including PMA, cycloheximide, AIF4, and okadaic acid. Although all of these agents stimulated c-Jun phosphorylation, cycloheximide and okadaic acid had no effect on pp42 MAP kinase phosphorylation, suggesting that MAP kinase activation was not necessary for c-Jun phosphorylation in vivo. Because dominant-negative RasAsn17 has been shown to block the effects of PMA on pp42 MAP kinase phosphorylation, we assessed its effect on c-Jun phosphorylation by cotransfection with a truncated c-Jun construct (c-Jun234). We found that c-Jun234 was expressed only in the cytosol and was inducibly phosphorylated with kinetics similar to those of endogenous nuclear c-Jun. Furthermore, we found that RasAsn17 had no effect on PMA-induced phosphorylation of c-Jun234. Because Ha-Ras requires isoprenylation for membrane binding, we examined the effect of the isoprenylation inhibitors lovastatin and perillic acid on PMA-induced c-Jun phosphorylation. Pretreatment of U937 cells with these agents had no effect on PMA-induced c-Jun or pp42 MAP kinase phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of differentiation and c-jun expression in human leukemic cells by okadaic acid, an inhibitor of protein phosphatases.

Okadaic acid, a protein phosphatase inhibitor, is a strong tumor promoter which activates protein phosphorylation. Because another activator of protein phosphorylation, phorbol esters, stimulates hematopoietic differentiation, we sought to determine whether okadaic acid could also induce the differentiation of the human leukemic cell line U937. Differentiation was assessed by measuring changes in the following: mRNA levels, cell growth, morphology, cell surface markers, and the ability to induce superoxide. We found that okadaic acid treatment of U937 cells induces immediate increases in total cellular levels of both c-jun and c-fos mRNAs. Nuclear run-on experiments demonstrate that initial increases are secondary to increases in transcription, whereas latter changes may be secondary to mRNA stabilization. Like phorbol esters, okadaic acid treatment also activates AP-1 enhancer activity and induces the phosphorylation of c-Jun protein. Approximately 6-12 hours after treatment with okadaic acid, mRNA levels of c-myc, p34cdc2, and p58GTA, two cell cycle regulated protein kinases, decrease. Okadaic acid inhibits the growth of U937 cells, induces changes in nuclear morphology, stimulates increases in Mac-1 and Leu 11 surface antigens, and induces these cells to produce superoxide. These changes, taken together, suggest that U937 cells have been induced by okadaic acid to differentiate towards a more mature cell type.

Upstream regions of the c-jun promoter regulate phorbol ester-induced transcription in U937 leukemic cells.

To understand the mechanism by which phorbol esters (PMA) stimulate c-jun transcription in human leukemic cell line U937, we have mutated specific enhancer sequences within the c-jun promoter. We find in the region of DNA from -132 to +170 containing Sp1, C-TF and AP-1 sequences that mutation of the AP-1 sequence alone is not sufficient to abrogate transcription, and mutation of the Sp1 sequence increases transcription 4-fold. Although mutation of the CTF site had no effect, CTF and AP-1 mutations together totally abrogate PMA-induced transcription. In comparison mutations of either of these sites alone or together in a construct containing -1639/+740 of the c-jun promoter had no effect on transcription. Because this data suggested the possibility of other upstream control regions, we sequenced the promoter from -142 to -1639. This sequence demonstrates a greater than 70% homology between human, and mouse c-jun promoters for the region from -142 to -441, and a second AP-1-like site in the -183 to -192 region. Mutation of this site did not influence transcription by PMA. By making constructs containing varying portions of the promoter, we have identified the region between -142 and -711 to be responsible for mediating PMA-induced c-jun transcription.

Expression of the human neutrophil elastase gene: positive and negative transcriptional elements in the 5' flanking region.

The structure and position of cis-acting DNA sequences which regulate tissue specific expression of the human neutrophil elastase (HNE) gene have been investigated. We have identified a positive and a negative regulatory element upstream from the promoter region. The ability of these sequences to regulate transcription in myeloid and non-myeloid cells was studied by inserting varying lengths of HNE 5'-flanking sequence into a reporter plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene. CAT activity in U937 was minimal in the absence of promoter and in the presence of HNE sequence to -102 bp. Inclusion of sequence up to -153 bp resulted in a 5.6-fold increase in CAT activity that was not observed in non-myeloid transfectants. Extension of the insert to include additional HNE sequence to -196 bp resulted in a decrease in CAT activity to control levels.

Regulation of c-jun expression and AP-1 enhancer activity by granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony stimulating factor (GM-CSF) stimulates the growth and differentiation of human hematopoietic progenitor cells by activating transcription of specific genes. The mechanism by which binding of GM-CSF to its receptor stimulates gene expression remains unknown. To examine this process in more detail, we have transfected human monocytic leukemia cells U937 with a plasmid containing an AP-1 enhancer element and a chloramphenicol acetyltransferase recorder gene and treated them with GM-CSF. We find that GM-CSF stimulates a 2-3-fold increase in chloramphenicol acetyltransferase activity over a concentration range 1-1,000 units/ml. Northern and Western blot analysis demonstrates that the mechanism by which GM-CSF stimulates AP-1 enhancer activity involves increases in c-jun and c-fos mRNA levels, and increases in Jun protein. In a similar fashion the treatment of normal human monocytes with GM-CSF also induced increases in total cellular c-jun. Because protein kinase C plays a crucial role in activating c-jun transcription we examined the role of this enzyme in mediating the effects of GM-CSF. Treatment of U937 cells with inhibitors of protein kinase C including staurosporine 10 nM and H-7 50 microM, or down-regulation of protein kinase C by phorbol ester pretreatment blocks the induction of c-jun by GM-CSF. However, HA which does not block protein kinase C had no effect on GM-CSF stimulation of c-jun RNA levels. In addition, GM-CSF treatment causes the rapid translocation of protein kinase C to the particulate fraction which was maximal by 5 min and returned to base line by 80 min. These data suggest that the binding of GM-CSF to its receptor stimulates increases in c-jun mRNA and protein and activates AP-1 enhancer activity. These effects may be at least in part mediated by activation of protein kinase C.