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PubMed1997

Sindbis virus replicons and Sindbis virus: assembly of chimeras and of particles deficient in virus RNA.

Alphaviruses are a well-characterized group of positive-strand RNA viruses. The identification of cis-acting elements in their genomes and their replication strategy have made them useful as vectors for the expression of heterologous genes. In infected cells, the nonstructural proteins, required for replication and transcription of the viral genes, are translated from the genomic RNA; the structural proteins, the capsid protein that interacts with the RNA to form the nucleocapsid and the proteins embedded in the lipid envelope, are translated from a subgenomic mRNA and can be replaced by heterologous genes. Such modified genomes are self-replicating (replicons); they can be introduced into the cells by transfection and can also be packaged into extracellular particles with defective helper (DH) RNAs. The particular DH RNA determines how well it is replicated and to what extent it is packaged. One potential complication of this system has been that recombination between the replicon genome and the DH RNA may occur. The studies described here were designed to prevent recombination by expressing the capsid protein from one DH RNA and the virus membrane proteins from a second helper RNA. Recombination to yield a nonsegmented infectious virus genome would then require several independent crossover events. There is a translational enhancer located downstream of the initiating AUG in the RNA of the capsid gene that had to be conserved in the second helper to achieve high-level expression of the viral glycoproteins. For this reason, we modified the capsid protein gene in two ways: the first was to use the capsid protein gene from a different alphavirus, Ross River virus, and the second was to make deletions in that gene to maintain the translational enhancer in the RNA but to eliminate the positively charged region in the protein that should be essential for the specific and nonspecific interactions with RNA. Transfections with replicon RNA and the deleted chimeric DH RNA as the only helper resulted in the high-level production of particles that were almost completely devoid of RNA. The inclusion of a helper expressing an intact Sindbis virus capsid protein gene led to the production of high levels of packaged replicons. Recombinants were not detected even after several undiluted passages.

Amino Acid Sequence↗

PubMed1999

A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo.

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, we have identified the cis-acting sequences required for this effect. We find that 1a-induced stabilization is mediated by a 150- to 190-base segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous beta-globin RNA. Within this intergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication.

Bromovirus↗

PubMed1995

Interferon treatment inhibits virus replication in HIV-1- and SIV-infected CD4+ T-cell lines by distinct mechanisms: evidence for decreased stability and aberrant processing of HIV-1 proteins.

We have examined the effects of interferon (IFN)-alpha/beta on HIV-1 and SIV replication in CD4+ T-cell lines. To enable us to examine these effects on a single cycle of virus replication, cells were synchronously infected with HIV-1 LAI or SIV mac251. Cell lines included MT4 cells which were responsive to IFN and, as controls, C8166 cells which failed to respond to interferon treatment. Similar to previous reports, we found that replication of both HIV-1 and SIV was markedly inhibited in responsive MT4 cell lines treated with IFN. No such decreases were observed in HIV-1-infected, IFN-treated C8166 cells. Levels of both intracellular and extracellular viral antigens decreased in both HIV-1- and SIV-infected MT4 cells treated with IFN. Whereas steady state levels of viral-specific RNAs dramatically declined in SIV-infected cells, no such decrease was observed in IFN-treated HIV-1-infected cells. In accordance with these data, the rate of viral protein synthesis did not significantly change in HIV-1-infected, IFN-treated MT-4 cells. Western blot analysis of extracts prepared from IFN-treated HIV-1-infected cells revealed a decreased accumulation of most HIV-1-specific glycoproteins and proteins with the exception of the pr55 gag precursor. Pulse-chase experiments confirmed the enhanced stability of pr55 in IFN-treated cells, but also unexpectedly demonstrated the accelerated and quantitative processing of the p26 precursor (p24 capsid [CA] plus p2) to the final processed p24 (CA) polypeptide. These data, taken together, suggest that IFN deregulated viral protein processing and caused reduced protein stability in HIV-1-infected cells while inhibiting an earlier stage of replication in SIV-infected cells.

Animals↗

PubMed2005

Detection of kappa and delta opioid receptors in skin--outside the nervous system.

Opioid receptors (OR) are widely expressed in the central nervous system (CNS). Opioid antinociception might be initiated by activation of OR outside the CNS, indicating targeting of peripheral OR could be useful in the treatment of chronic pain. This study was designed to detect OR in skin tissues of healthy volunteers at both mRNA and protein levels. Skin samples from 10 healthy individuals were investigated. Total isolated RNAs were reverse transcribed, amplified and quantified by real-time PCR. Tissue and skin fibroblast OR protein was detected by immunohistochemistry, Western blot, and immunofluorescence. All skin tissue samples expressed delta- (DOR) and kappa-OR (KOR) mRNAs. Using immunohistochemistry, DOR and KOR were localized in skin fibroblast-like and mononuclear cells. Skin fibroblasts in culture expressed DOR and KOR mRNA. Using immunofluorescence, both DOR and KOR proteins were expressed predominantly on the cell membrane with minor staining in the cytoplasm. We suggest that enhanced expression of DOR and KOR in skin justifies the exploration of selective novel delta and kappa agonists for local pain treatment.

Adult↗

PubMed2000

A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures.

DsrA is an 87-nucleotide regulatory RNA of Escherichia coli that acts in trans by RNA-RNA interactions with two different mRNAs, hns and rpoS. DsrA has opposite effects on these transcriptional regulators. H-NS levels decrease, whereas RpoS (final sigma(s)) levels increase. Here we show that DsrA enhances hns mRNA turnover yet stabilizes rpoS mRNA, either directly or via effects on translation. Computational and RNA footprinting approaches led to a refined structure for DsrA, and a model in which DsrA interacts with the hns mRNA start and stop codon regions to form a coaxial stack. Analogous bipartite interactions exist in eukaryotes, albeit with different regulatory consequences. In contrast, DsrA base pairs in discrete fashion with the rpoS RNA translational operator. Thus, different structural configurations for DsrA lead to opposite regulatory consequences for target RNAs.

Bacterial Proteins↗

PubMed2003

Suppression of hepatitis B virus core promoter by the nuclear orphan receptor TR4.

The TR4 orphan receptor is a member of the nuclear receptor superfamily that modulates gene expression via binding to the AGGTCA direct repeat hormone response element. Here we report a functional study of TR4 interaction with the core promoter of the hepatitis B virus (HBV). The electrophoretic mobility shift assay shows that TR4 can bind to the direct repeat 1 sequence element (AGGTTAAAGGTCT, nucleotide coordinates 1757-1769, TR4RE-HBV) on the HBV core promoter. TR4 also can enhance the activity of a synthetic luciferase reporter linked with four copies of TR4RE-HBV in either liver HepG2 or non-liver H1299 cells in a dose-dependent manner. Surprisingly, TR4 represses the activity of a luciferase reporter containing the entire HBV genome sequences. Moreover, mutation of this TR4RE-HBV site in the HBV core promoter diminishes the TR4 suppression effect. This TR4-induced suppression of HBV core promoter activity is further confirmed by primer extension analysis of the HBV core RNAs, showing that TR4 represses both pre-core and core mRNAs. Further dissection of this repressive mechanism indicates that TR4 may suppress the HBV core promoter activity via repressing HNF4alpha-mediated transactivation by protein-protein interactions without inhibition of HNF4alpha DNA binding. Furthermore, our results indicate that the N- and C-terminal regions of TR4 protein are required for TR4-HNF4alpha interaction. It is possible that TR4-HNF4alpha interaction may block the HNF4alpha function that results in the suppression of HBV gene expression. Together, these results demonstrate that TR4 can serve as a negative modulator in the transcriptional regulation of HBV core gene expression.

Binding Sites↗

PubMed1977

Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine.

An in vitro transcription system in which vesicular stomatitis virus (VSV) mRNA species have been synthesized is described. In addition to purified VSV virions, which contain an RNA-dependent RNA polymerase, this system contained a cytoplasmic cell extract that enhanced correct transcription. Gel electrophoretic analysis of the methylated polyadenylic acid [poly(A)]-containing VSV mRNA produced in this system in the presenct of S-adenosylmethionine showed the discrete VSV mRNA species. However, when unmethylated mRNA was synthesized in the presence of S-adenosylhomocysteine, the poly(A)-containing transcripts were large and heterogeneous in molecular weight and did not contain discrete VSV mRNA species. Two-dimensional fingerprint analysis of the methylated and unmethylated products suggested that identical nucleotide sequences were present in the RNAs. Further analysis showed the presence of very large heterogeneous poly(A), 200 to 2,000 nucleotides in lenght, in the unmethylated transcript. Proof that this large poly(A) was covalently linked to the correct VSV mRNA transcripts was obtained by removal of the poly(A) by hybirdization with oligodeoxythymidylic acid and digestion with RNase H. This digestion produced unmethylated VSV mRNA transcripts with the same discrete sizes as the deadenylated RNAs produced from VSV mRNA initially isolated from VSV-infected cells. The results suggest that there is a relationship between methylation at the 5'-end and polyadenylation at the 3'-end of VSV mRNA's. Furthermore, addition of the very large poly(A) does not affect the normal process of sequential transcription of the VSV genome, suggesting that this poly(A) addition is occurring independently of further transcription.

Cell-Free System↗

PubMed1998

Enhanced expression of metabotropic glutamate receptor 3 messenger RNA in the rat spinal cord during ultraviolet irradiation induced peripheral inflammation.

Metabotropic glutamate receptors are thought to play a role in the development and maintenance of spinal hyperexcitability resulting in hyperalgesia and pain. In this study we have used in situ hybridization to investigate the distribution of metabotropic glutamate receptors mGluR1-7 messenger RNA in the rat spinal cord in a model of inflammatory hyperalgesia. Hyperalgesia was induced in nine-day-old rats by exposure of the left hindpaw to an ultraviolet light source. Lumbar portions of spinal cords were removed from control and ultraviolet-treated animals. In situ hybridization with specific oligonucleotide probes was used to localize metabotropic glutamate receptor messenger RNAs. mGluR1, 3-5 and 7 subtype messenger RNA was detected in the gray matter of the spinal cord with distribution being specific for the different subtypes. A significant increase in the expression of mGluR3 messenger RNA was seen in cells of the dorsal laminae in both sides of the lumbar spinal cord. This increase was most pronounced in laminae II, III and IV but gradually decreased and disappeared by the third day of inflammation. In parallel with this, behavioural experiments revealed mechanical hyperalgesia in both hindlimbs after ultraviolet irradiation. There was no change in mGluR3 messenger RNA expression in the thoracic segments. No changes have been detected in the levels of expression of mGluR 1,2,4,5,7 subtype messenger RNA in spinal cords taken from hyperalgesic animals. These observations show that during ultraviolet irradiation induced inflammation, the synthesis of mGluR3 messenger RNA is altered suggesting that regulation of metabotropic glutamate receptor expression may be instrumental in plastic changes within the spinal cord during the development of hyperalgesia and pain.

Amino Acid Sequence↗

PubMed2003

Kinesin I and cytoplasmic dynein orchestrate glucose-stimulated insulin-containing vesicle movements in clonal MIN6 beta-cells.

Glucose-stimulated mobilization of large dense-core vesicles (LDCVs) to the plasma membrane is essential for sustained insulin secretion. At present, the cytoskeletal structures and molecular motors involved in vesicle trafficking in beta-cells are poorly defined. Here, we describe simultaneous imaging of enhanced green fluorescent protein (EGFP)-tagged LDCVs and microtubules in beta-cells. Microtubules exist as a tangled array, along which vesicles describe complex directional movements. Whilst LDCVs frequently changed direction, implying the involvement of both plus- and minus-end directed motors, inactivation of the minus-end motor, cytoplasmic dynein, inhibited only a small fraction of all vesicle movements which were involved in vesicle recovery after glucose-stimulated exocytosis. By contrast, selective silencing of the plus-end motor, kinesin I, with small interfering RNAs substantially inhibited all vesicle movements. We conclude that the majority of LDCV transport in beta-cells is mediated by kinesin I, whilst dynein probably contributes to the recovery of vesicles after rapid kiss-and-run exocytosis.

Animals↗

PubMed1997

The dynamic NMR structure of the T psi C-loop: implications for the specificity of tRNA methylation.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependent methylation of uridine-54 in the T psi C-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried and the question arises as to how RUMT gains access to the methylation site. A 17-mer RNA hairpin consisting of nucleotides 49-65 of the T psi-loop is a substrate for RUMT. Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD) methods were used to determine the solution structure of the 17-mer T-arm fragment. The loop of the hairpin exhibits enhanced flexibility which renders the conventional NMR average structure less useful compared to the more commonly found situation where a molecule exists in predominantly one major conformation. However, when resorting to softer refinement methods such as MD with time-averaged restraints, the conflicting restraints in the loop can be satisfied much better. The dynamic structure of the T-arm is represented as an ensemble of 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the T psi-loop in solution in conjunction with extensive binding studies of RUMT with the T psi C-loop and tRNA suggest that the specificity of the RUMT/ tRNA recognition is associated with tRNA tertiary structure elements. For the methylation, RUMT would simply have to break the tertiary interactions between the D- and T-loops, leading to a melting of the T-arm structure and making U54 available for methylation.

Base Sequence↗

PubMed2002

Characterization of Tbc2, a nucleus-encoded factor specifically required for translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii.

Genetic analysis has revealed that the three nucleus-encoded factors Tbc1, Tbc2, and Tbc3 are involved in the translation of the chloroplast psbC mRNA of the eukaryotic green alga Chlamydomonas reinhardtii. In this study we report the isolation and phenotypic characterization of two new tbc2 mutant alleles and their use for cloning and characterizing the Tbc2 gene by genomic complementation. TBC2 encodes a protein of 1,115 residues containing nine copies of a novel degenerate 38-40 amino acid repeat with a quasiconserved PPPEW motif near its COOH-terminal end. The middle part of the Tbc2 protein displays partial amino acid sequence identity with Crp1, a protein from Zea mays that is implicated in the processing and translation of the chloroplast petA and petD RNAs. The Tbc2 protein is enriched in chloroplast stromal subfractions and is associated with a 400-kD protein complex that appears to play a role in the translation of specifically the psbC mRNA.

Alleles↗

PubMed2003

Inhibition of HCV NS3 protease by RNA aptamers in cells.

Non-structural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct activities, protease and helicase, which are essential for HCV proliferation. In previous work, we obtained RNA aptamers (G9-I, II and III) which specifically bound the NS3 protease domain (DeltaNS3), efficiently inhibiting protease activity in vitro. To utilize these aptamers in vivo, we constructed a G9 aptamer expression system in cultured cells, using the cytomegarovirus enhancer + chicken beta-actin globin (CAG) promoter. By conjugating the cis-acting genomic human hepatitis delta virus (HDV) ribozyme and G9-II aptamer, a chimeric HDV ribozyme-G9-II aptamer (HA) was constructed, which was used to produce stable RNA in vivo and to create tandem repeats of the functional unit. To target the transcribed RNA aptamers to the cytoplasm, the minimal mutant of constitutive transport element (CTE), derived from type D retroviruses, was conjugated at the 3' end of HA (HAC). Transcript RNAs from (HA)(n) and (HAC)(n) were processed into the G9-II aptamer unit by the cis-acting HDV ribozyme, both in vitro and in vivo. Efficient protease inhibition activity of HDV ribozyme-G9-II aptamer expression plasmid was demonstrated in HeLa cells. Protease inhibition activity level of tandem chimeric aptamers, (HA)(n) and (HAC)(n), rose with the increase of n from 1 to 4.

Base Sequence↗

PubMed1990

Active site labeling of Escherichia coli transcription elongation complexes with 5-[4-azidophenacyl)thio)uridine 5'-triphosphate.

Escherichia coli RNA polymerase transcription elongation complexes have been prepared that contain a photo-cross-linking uridine analog at only the 3' end, or one or two nucleotides removed from the 3' end, in the nascent RNA chain. Additionally, complexes have been isolated in which the analog has been substituted for every UMP residue, at positions ranging from 20 to 140 nucleotides from the 3' end. The RNA has been photochemically cross-linked to the RNA polymerase to identify the subunits that form the binding site(s) for these regions in the nascent RNA. The photo-cross-linking nucleotide analog used for these studies was 5-[4-azidophenacyl)thio)uridine-5'-triphosphate (5-APAS-UTP), which acts as a 10-15 A probe. With 5-APAS-UMP positioned only at the 3' end of the RNA, or one or two nucleotides from the 3' end, only the beta subunit appeared to be contacted. When the analog was positioned throughout the RNA, both the beta and beta' subunits were contacted. No labeling of the sigma or alpha subunits was observed with any RNA length. In addition to placing this analog at specific positions in short RNAs, we have carried out transcription studies with 5-APAS-UTP to determine the optimal UTP to analog ratio for production of full length, photoreactive transcripts. Surprisingly, we found that when transcription complexes were stalled shortly after initiation, by deletion of one ribonucleoside triphosphate to synchronize transcription, changes in transcriptional pausing occurred downstream. These results suggest that events that occur early in transcription can affect the elongation and/or termination events that occur far downstream from the promoter. This effect occurred even with UTP but was greatly enhanced by replacement of UTP with either this analog or 4-thio-UTP. By enhancing the normal transcriptional pausing event, these analogs can serve as probes of the conformational changes that may exist in paused transcription complexes.

Affinity Labels↗

PubMed1990

Genetic engineering of plants for virus resistance.

Historically, control of plant virus disease has involved numerous strategies which have often been combined to provide effective durable resistance in the field. In recent years, the dramatic advances obtained in plant molecular virology have enhanced our understanding of viral genome organizations and gene functions. Moreover, genetic engineering of plants for virus resistance has recently provided promising additional strategies for control of virus disease. At present, the most promising of these has been the expression of coat-protein coding sequences in plants transformed with a coat protein gene. Other potential methods include the expression of anti-sense viral transcripts in transgenic plants, the application of artificial anti-sense mediated gene regulation to viral systems, and the expression of viral satellite RNAs, RNAs with endoribonuclease activity, antiviral antibody genes, or human interferon genes in plants.

Base Sequence↗

PubMed2003

Evolution of chromatin-remodeling complexes: comparative genomics reveals the ancient origin of "novel" compensasome genes.

Dosage compensation in Drosophila is mediated by a complex, called compensasome, composed of at least five proteins and two noncoding RNAs. Genes encoding compensasome proteins have been collectively named male-specific lethals or msls. Recent work showed that three of the Drosophila msls (msl-3, mof, and mle) have an ancient origin. In this study, I describe likely orthologues of the two remaining msls, msl-1 and msl-2, in several invertebrates and vertebrates. The MSL-2 protein is the only one found in Drosophila and vertebrate genomes that contains both a RING finger and a peculiar type of CXC domain, related to the one present in Enhancer of Zeste proteins. MSL-1 also contains two evolutionarily conserved domains: a leucine zipper and a second characteristic region, described here for the first time, which I have called the PEHE domain. These two domains are present in the likely orthologues of MSL-1 as well as in other genes in several invertebrate and vertebrate species. Although it cannot be excluded that the compensasome complex is a recent evolutionary novelty, these results shows that all msls are found in mammals, suggesting that protein complexes related to the compensasome may be present in mammalian species. Metazoans that lack several of the msls, such as Caenorhabditis elegans, cannot contain compensasomes. The evolutionary relationships of the compensasome and the NuA4 complex, another chromatin-remodeling complex that contains related subunits, are discussed.

Amino Acid Motifs↗

PubMed1998

Generation of replication-competent hepatitis B virus nucleocapsids in insect cells.

The double-stranded DNA genome of human hepatitis B virus (HBV) and related hepadnaviruses is reverse transcribed from a pregenomic RNA by a viral polymerase (Pol) harboring both priming and RNA- and DNA-dependent elongation activities. Although hepadnavirus replication occurs inside viral nucleocapsids, or cores, biochemical systems for analyzing this reaction are currently limited to unencapsidated Pols expressed in heterologous systems. Here, we describe cis and trans classes of replicative HBV cores, produced in the recombinant baculovirus system via coexpression of HBV core and Pol proteins from either a single RNA (i.e., in cis) or two distinct RNAs (in trans). Upon isolation from insect cells, cis and trans cores contained Pol-linked HBV minus-strand DNA with 5' ends mapping to the authentic elongation origin DR1 and also plus-strand DNA species. Only trans cores, however, were highly active for the de novo priming and reverse transcription of authentic HBV minus strands in in vitro endogenous polymerase assays. This reaction strictly required HBV Pol but not the epsilon stem-loop element, although the presence of one epsilon, or better, two epsilons, enhanced minus-strand synthesis up to 10-fold. Compared to unencapsidated Pol enzymes, encapsidated Pol appeared to be (i) highly processive, able to extend minus-strand DNAs of 400 nucleotides from DR1 in vitro, and (ii) more active for HBV plus-strand synthesis. These observations suggest possible contributions to the replication process from the HBV core protein. These novel core reagents should facilitate the analysis of HBV replication in its natural environment, the interior of the capsid, and also fuel the development of new anti-HBV drug screens.

Animals↗

PubMed1995

Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors.

Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.

Amino Acid Sequence↗

PubMed1996

Increases in HSF1 translocation and synthesis in human epidermoid A-431 cells: role of protein kinase C and [Ca2+]i.

BACKGROUND: It is known that heat shock increases both heat shock protein 70 kd (HSP-70) mRNA synthesis, and intracellular cytosolic free calcium concentration ([Ca2+]i). The latter enhances the heat inducible form of HSP-70 production by increasing the complex formation between heat shock transcriptional factor (HSF) and heat shock elements (HSE). In this study, we investigated the effect of agonists (PMA; ionomycin) and antagonists (BAPTA; staurosporine) of protein kinase C (PKC), and calcium channel on translocation and synthesis of HSF1, and activation of HSP-70 gene in human epidermoid A-431 cells. METHODS: Cells were incubated with poly 12-myristate 13-acetate (PMA) or ionomycin at different concentrations for various periods of time. Messenger RNAs of HSF and HSP-70 were measured with RT-PCR. The HSP-70 protein was determined with Western blots, and HSF protein was measured by gel mobility retardation assay. RESULTS: Significant increases in HSF binding to [32P]labeled HSE were found at 30 minutes in nuclear extract and at 4 hours in both nuclear and cytosol extracts. The PMA- and ionomycin-induced increases in HSF were in a concentration-dependent manner with a maximal increase at 10(-6) mol/L of each drug. Meanwhile, the mRNAs encoded for HSF1 and HSP-70, but not HSF2, were significantly increased and reached the maximum at 1 hour after the treatment. The PMA increased [Ca2+]i by 92% because of Ca2+ influx. The increases in mRNA of HSF1 and HSP-70 induced by treatment with 1 mumol/L PMA were completely blocked by preincubating cells with either 2 mumol/L staurosporine in the presence of extracellular Ca2+ or 100 mumol/L BAPTA-am in absence of extracellular Ca2+. Like PMA, the increases induced by ionomycin were also inhibited by 100 mumol/L BAPTA-am in absence of extracellular Ca2+. Furthermore, Western blots show that 1 mumol/L PMA or ionomycin induced maximal increase in HSP-70 after 7 hours of continuous incubation with either agent. When cells were simultaneously treated with 1 mumol/L PMA and ionomycin together for 1 hour, the increase in HSP-70 and HSF1 mRNAs reached a greater level than the level stimulated by either drug alone. CONCLUSIONS: These results indicate that both PMA and ionomycin stimulate HSF1, but not HSF2, translocation and synthesis leading to the HSP-70 expression and that their effects are Ca(2+)-dependent.

Calcium↗