Increase In Rate Of Transcription Research Articles

Activation of basal transcription by a mutation in SIN4, a yeast global repressor, occurs through a mechanism different from activator-mediated transcriptional enhancement.

The Saccharomyces cerevisiae protein Sin4 has been suggested to affect the transcription of various genes by locally altering chromatin structure. Previous studies have defined two classes of promoters: those which are activated by loss of SIN4 function (termed sin4-responsive promoters) and those which are not activated by sin4 mutations (termed sin4 non-responsive promoters). We analyzed the mechanism of this differential response of the two classes of promoters to a sin4 mutation. The sin4 non-responsive promoters were activated when upstream elements in the promoter region were eliminated. The upstream elements of sin4 non-responsive promoters were, in turn, found to repress the activity of the sin4-responsive promoters in an orientation-independent manner. The sin4-mediated activation was repressed by the Rme1- but not by the Tup1-Ssn6-mediated repression system. Activation of sin4-responsive promoters by Pho4 and the sin4 mutation was additive, and enhancement of transcription driven by sin4-responsive promoters was found to be due to an increase in the basal rate of transcription. The upstream regions in the sin4 non-responsive promoters contained elements that were able to inhibit activation of basal transcription. Based on these observations, we suggest that activation of basal transcription by a mutation in a gene for a global repressor, SIN4, occurs through a mechanism that differs from that responsible for activator-mediated transcriptional enhancement, and we therefore propose that basal transcription and activator-mediated transcription are repressed by different mechanisms.

Onset of zygotic transcription and maternal transcript legacy in the rabbit embryo.

Onset of zygotic transcription is progressive from the one-cell stage onward in the rabbit embryo. Maternal transcripts remain fairly stable until the 8-16 cell stage when major transcriptional activation of the zygotic genome takes place. To understand the mechanisms of the maternal-to-zygotic transition in the genetic information governing development, we asked whether a progressive synthesis of zygotic transcripts takes over the maternal molecules, or whether the synthesis of zygotic transcripts is very abrupt and independent of the persistence of the maternal counterparts. To answer this question, we set up mRNA differential display experiments comparing the mRNA content of rabbit embryos at different stages during the preimplantation period. We isolated eight zygotic transcripts whose synthesis is abruptly turned on at the 8-16 cell stage. These transcripts are involved in general cellular metabolism and their maternal counterparts are still present up to the four-cell and even the 8-16 cell stage. This identification of early zygotic transcripts suggests that global long range modifications of chromatin structure result in a rapid increase in transcription rates during the major transcriptional activation of the zygotic genome.

Oxygen stress increases prolyl cis/trans isomerase activity and expression of cyclophilin 18 in rabbit blastocysts.

The peptidyl-prolyl cis/trans isomerase (PPIase) activity and the expression of cyclophilins were studied in 6-day-old rabbit preimplantation embryos cultured under physiological and increased oxygen concentrations of 5% and 20% O(2), respectively. The PPIase activity was completely inhibited by cyclosporin A (CsA). The inhibitor of FK506-binding proteins, rapamycin, had no effect on the PPIase activity, indicating that the PPIase activity in rabbit blastocysts originates from cyclophilins. Using CsA affinity chromatography, only one cyclophilin with a molecular mass of about 17.8 kDa was separated. The cDNA of rabbit cyclophilin was cloned and sequenced. Analysis of the 682-base pair cDNA revealed an open reading frame coding for a polypeptide of 164 amino acid residues with a molecular weight of 17.83 kDa. Homologies of 90% and 96% for the cDNA and amino acid sequence, respectively, to the human CyP18 were found, suggesting that the novel rabbit cyclophilin is a member of the CyP18 family (rabCyP18). The transcription level of rabCyP18 mRNA was 8.3 +/- 0.6 pg in 100 ng total RNA in noncultured blastocysts. In vitro culture with moderate oxygen stress (20% O(2)) resulted in a 1.5-fold increase in rabCyP18 transcription and an increased PPIase activity compared to that of blastocysts cultured with 5% O(2). Increase in transcription rate and PPIase activity by oxygen stress suggests an involvement of CyP18 in oxygen defense in rabbit preimplantation embryos.

Exercise attenuates nuclear protein binding to gene regulatory sequences of hepatic fatty acid synthase.

The effect of an acute bout of exhaustive exercise on hepatic fatty acid synthase (FAS) gene expression was examined in rats. Female Sprague-Dawley rats (age 8 wk) were fasted for 48 h (F, n = 6), or fasted, refed a high-fructose diet for 6 h, and killed at rest (R, n = 6) or killed after running on a treadmill at 27 m/min and 5% grade for 88 +/- 7 min (E, n = 6). Gel mobility shift assay indicated that R rats had twofold higher liver nuclear protein binding to oligonucleotides corresponding to the insulin responsive sequence (-71/-50) and carbohydrate response element (+283/+303) on the FAS promoter, compared with F rats. Exercise severely attenuated this binding in liver nuclear extracts to the levels seen in F rats. Competition and supershift experiments revealed that the bound protein complexes contained the upstream stimulatory factors. Nuclear run-on experiment revealed a 49-fold increase in transcription rate of the FAS gene in R vs. F rats, whereas exercise suppressed the transcription rate. FAS mRNA abundance and FAS enzyme activity were dramatically increased with refeeding but were unaltered by exercise. The results reveal that dietary induction of hepatic FAS is stimulated by increased nuclear protein binding to insulin responsive sequence and carbohydrate response element, whereas exhaustive exercise attenuates the binding, which may precede downregulation of FAS mRNA and enzyme synthesis reported in our previous work (M. A. Griffiths, R. Fiebig, M. T. Gore, D. H. Baker, K. Esser, L. Oscai, and L. L. Ji. J. Nutr. 126, 1959-1971, 1996).

Multiple mechanisms control the expression of abscisic acid (ABA)‐requiring genes in tomato plants exposed to soil water deficit

ABSTRACTElevated levels of abscisic acid (ABA) are required for the expression of several genes during water deficit. The expression patterns of the ABA‐requiring genes, le4, le16, le25 and his1‐s, differed during a 4 d period of water deficit and a 24 h recovery period in intact tomato plants. During water deficit, a strong correlation between ABA content and mRNA accumulation was observed for le16, with only a moderate or poor correlation observed between the accumulation of ABA and the other mRNAs. The regulation of le4 and le16 can be accounted for by an increase in transcription rate, while components of post‐transcriptional regulation are implicated in the regulation of the le25 and his1‐s genes. A strong correlation between leaf ABA content and the transcription rate of le4 and le16 during the stress and recovery periods was observed. Protein synthesis was not necessary for the expression of the four genes. However, the mRNA content of le25 increased in the presence of translation inhibitors plus ABA, implicating inhibition of a transcriptional repressor. The regulation of gene expression in response to stress is complex; although ABA was required for the regulation of the genes in this study, there must be additional control mechanisms that interact with components of the ABA regulatory machinery.

Antiterminator-dependent modulation of transcription elongation rates by NusB and NusG.

Ribosomal RNA is transcribed about twice as fast as messenger RNA in vivo, and this increased transcription rate requires the rrn boxA antitermination system. Because several Nus factors have been implicated in rrn antitermination, we have examined the role of NusB, NusE and NusG in controlling the rate of rrn boxA-mediated transcript elongation. In vivo RNA polymerase transcription rates were determined by measuring the rate of appearance of lacZ transcript using a plasmid that contained an inducible T7 promoter fused to the rrn boxA sequence followed by the lacZ gene. This plasmid was introduced into Escherichia coli mutant strains that can be conditionally depleted of NusG, or that carry a deficient nusB gene or a nusE mutation. We found that, in addition to the rrn boxA antiterminator sequence, both NusG and NusB were required to maintain the high transcription rate. The nusE mutation used in this study may be specific for lambda antitermination, as it did not influence the boxA-mediated increase in transcription rate.

Hypoxia and Vascular Endothelial Growth Factor Selectively Up-regulate Angiopoietin-2 in Bovine Microvascular Endothelial Cells

Recent studies have shown that the angiopoietin-Tie2 system is a predominant regulator of vascular integrity. In this study, we investigated the effect of two known angiogenic stimuli, hypoxia and vascular endothelial growth factor (VEGF), on these molecules. VEGF induced both a time- and concentration-dependent increase in angiopoietin-2 (Ang2) mRNA expression in bovine microvascular endothelial cells. This up-regulation was derived primarily from an increased transcription rate as evidenced by nuclear run-on assay and mRNA decay study. The increased Ang2 expression upon VEGF treatment was almost totally abolished by inhibition of tyrosine kinase or mitogen-activated protein kinase and partially by suppression of protein kinase C. Hypoxia also directly increased Ang2 mRNA expression. In contrast, Ang1 and Tie2 responded to neither of these stimuli. The enhanced Ang2 expression following VEGF stimulation and hypoxia was accompanied by de novo protein synthesis as detected by immunoprecipitation. In a mouse model of ischemia-induced retinal neovascularization, Ang2 mRNA was up-regulated in the ischemic inner retinal layer, and remarkable expression was observed in neovascular vessels. These data suggest that both hypoxia- and VEGF-induced neovascularization might be facilitated by selective induction of Ang2, which deteriorates the integrity of preexisting vasculature.

UV irradiation upregulates Egr-1 expression at transcription level.

UVC irradiation rapidly and strongly induces protein expression of the early growth response-1 gene (Egr-1) encoding a transcription factor which may have a protective function against UV damage. In this paper, we further investigate mechanisms responsible for such induction. We show that UVC irradiation also induced Egr-1 mRNA expression, increased transcription rate by nuclear run-on assay and stimulated Egr-1 promoter activity by CAT assay. The Egr-1 mRNA stability remained unchanged in UVC-treated cells. On the other hand, UVC irradiation slightly extended Egr-1 protein half-life. The induction of Egr-1 by UVC was observed in many different cell types. UVA and UVB also strongly induced Egr-1 expression. These results indicate that UVC regulates Egr-1 expression at transcription level. The induction pattern of Egr-1 by UV suggests the importance of Egr-1 in the UV response.

Stage-specific regulation of stem cell factor gene expression in the rat seminiferous epithelium.

To assess the regulation of stem factor factor (SCF) gene expression during spermatogenesis, we tested the effects of hormones (FSH, testosterone, and 17beta-estradiol) and some growth factors [transforming growth factor-beta (TGF beta), TGF alpha, tumor necrosis factor-alpha, and activin] on SCF gene expression by using a transillumination-assisted microdisection technique, a seminiferous tubule culture system, and Northern hybridization. Our results showed that FSH (10 ng/ml) increased steady state levels of SCF messenger RNA (mRNA) in a stage-specific and time-dependent manner. 8-Bromo-cAMP could increase the SCF mRNA level in a similar way as FSH, whereas phorbol 12-myristate 13-acetate had no effect. Actinomycin D could abolish the stimulatory effect of FSH, whereas cyclohexamide could not. The half-life of SCF mRNA was apparently prolonged after FSH stimulation (FSH-treated tubules, 15.6 +/- 1.2 h; controls, 8.6 +/- 2.7 h). Nuclear run-on assay revealed 5- and 10-fold increases in the transcription rate after FSH stimulation for 8 and 30 h, respectively. Neither testosterone nor estradiol had significant effects on SCF gene expression in our tissue culture system. Activin, TGF beta, TGF alpha, and tumor necrosis factor-alpha had no effect on SCF gene expression in vitro. In conclusion, SCF gene expression in the rat seminiferous tubule is regulated by FSH through the cAMP/protein kinase A pathway. FSH regulates SCF gene expression at both transcriptional and posttranscriptional levels involving the increase in transcription rate and prolongation of half-life of SCF mRNA, but is independent of de novo protein synthesis.

Prolactin and its receptor in human endometrium.

Synthesis of prolactin (PRL) in human endometrium extends from the late luteal phase of the menstrual cycle throughout the pregnancy. We have studied the hormonal requirements for the sustained production of PRL and its receptor (PRL-R) in a long-term primary cell culture system. Progestin stimulates the production PRL and its receptor when stromal cells transform into decidual cells. The rise in PRL production rate correlates with an increase in steady-state PRL mRNA levels which are caused by increased transcription rate gene. Replacing progestin by the antiprogestin, RU 486, causes a transient superinduction of PRL production followed by reduction to basal level of expression. On the other hand, RU 486 exerts immediate inhibition of PRL-receptor mRNA expression. In addition, relaxin (RLX) enhances PRL synthesis. The transcription of the PRL gene in endometrium is dependent upon the promotor 6-kb upstream of the transcription start site in the pituitary. That biological functions of PRL and its receptor are critical to implantation and the maintenance of pregnancy is suggested by the impaired fertility of PRL and PRL-R knockout mice. PRL enhances endometrial cell growth at low concentrations and inhibits it at high concentrations. This dual action indicates an autocrine action of PRL-R-mediated signaling transduction pathways during reproductive cycles and pregnancy. During gestation, decidual-derived prolactin regulates the volume of amniotic and fetal extracellular fluid and electrolytes.

Hepatocyte growth factor in transgenic mice.

In order to clarify the function of hepatocyte growth factor (HGF) in vivo, we have developed transgenic mice expressing HGF in the liver. The bromodeoxyuridine labelling indices in livers from HGF transgenic mice were doubled, compared to those from wild type mice. Livers of HGF transgenic mice expressed high levels of c-myc, which was the consequence of increased transcription rates through the c-myc promoter. After 70% partial hepatectomy, the livers of HGF transgenic mice recovered in half the time needed for their normal siblings. Since we found that HGF inhibits growth of hepatocellular carcinoma (HCC) cells in vitro, we have made two kinds of double transgenic mice: HGF/TGF alpha and HGF/c-myc mice. The double transgenic mice expressing both HGF and TGF alpha had lower tumour yields, compared to TGF alpha transgenic mice. The HGF/c-myc double transgenic mice had a lower incidence of hepatocellular adenoma (HCA) and HCC in comparison with c-myc transgenic mice. In HGF/c-myc mice, there were more apoptotic cells and less mitotic cells than c-myc transgenic mice. These data indicate that HGF inhibits growth and occurrence of HCC in vivo. We also found that HGF protects liver from D-galactosamine (D-GalN)-induced injury. Hepatic prostaglandin E 2 (PGE2) contents in HGF transgenic mice were much higher than those in wild type mice, and were associated with hepatic HGF contents. An anti-HGF antibody inhibits production of PGE2 in liver after D-GalN administration. These data suggest that HGF protects liver from D-GalN-induced injury through increased liver PGE2 production. The data obtained from HGF transgenic mice suggests the possibility that HGF could be applicable for therapy of human liver diseases in the future.

Translational Control of MHC Class II I-A Molecules by IFN-γ

AbstractMHC class II molecules are expressed in a limited number of cell types, including B lymphocytes and macrophages (Mφ). IFN-γ increases the surface expression of class II molecules in a murine B cell line without inducing detectable changes in either I-A or I-A mRNA levels. In bone marrow-derived Mφ, IFN-γ causes an increase in class II expression at both the mRNA and surface levels. In addition to the increase in transcription rates described for Mφ, IFN-γ increases the rate of synthesis of IAα and IAβ proteins and the ribosome loading for both mRNA molecules in both cell types. Interestingly, there is a significant peak of free I-A mRNA in noninduced cells. Therefore, IFN-γ regulates the expression of MHC class II molecules at the translational level in both B cells and Mφ and, as already reported, at the transcriptional level only in Mφ. The actual mechanism of regulation causes changes in the translation initiation rates in both cell types, as demonstrated by an increase in ribosome loading in polysome gradients.

Posttranscriptional Lipopolysaccharide Regulation of the Lysozyme Gene at Processing of the Primary Transcript in Myelomonocytic HD11 Cells

Lysozyme is increasingly expressed in macrophages in inflammatory response to bacterial LPS. In this study, we investigated the mechanisms that control expression of the lysozyme gene in myelomonocytic HD11 cells activated by LPS. Nuclear run-on transcription assays showed that LPS caused a 15-fold increase in the transcription rate of the lysozyme gene. However, Northern analyses with lysozyme cDNA and intron sequences revealed that the LPS-induced increase in nuclear lysozyme transcripts greatly exceeded the increase in transcription rate. Furthermore, nuclear lysozyme transcripts in untreated cells with a t(1/2) of <10 min were more unstable than those accumulated in LPS-activated cells. We suggested, therefore, that the increased lysozyme expression following LPS treatment was largely due to a nuclear stabilization of the primary transcript. Interestingly, the increase in stability of the lysozyme primary transcript was accompanied by changes in nuclear processing including an increase in poly(A) tail length, which gradually shortened after entering the cytoplasm. The long lysozyme poly(A) tail, however, did not result in any increase in polysomal recruitment for translation or in stability of the cytoplasmic lysozyme mRNA.

A Novel NF-κB Enhancer Region Regulates Cytokine Induction Of The Human Inducible Nitric Oxide Synthase (iNOS) Gene Promoter

123 Nitric oxide (NO) is a potent immunomodulator which has diverse roles during infection, inflammation, and solid organ transplant rejection. Previously, we have shown that a combination of cytokines stimulate human inducible NO synthase (iNOS) expression, however, almost nothing is known about the specific cytokine-activated nuclear factors that control iNOS gene transcription. To determine the molecular mechanisms and transcription factors regulating expression of the human iNOS gene, we cloned and sequenced 7.2 kilobases (kb) of the gene's 5′-flanking DNA. To characterize this region, we generated deletional constructs of the human iNOS promoter (1.3 to 7.2 kb) linked to the luciferase reporter gene. The deletional DNA promoter constructs were transfected into human liver or lung epithelial cells using liposomes and then stimulated with cytokine mix (CM) of TNFα (500 U/ml)+ IL-1β (100 U/ml) + IFNγ (250 U/ml). Cell extracts were assayed for luciferase activity as a measure of iNOS promoter strength (graph, mean± SEM, n=6 per group): No increase in luciferase activity was noted with the 1.3 or 4.7 kb iNOS promoter constructs in response to CM. However, in both cell types a 3 and 5-fold increase in promoter activity was seen with the 5.8 kb and 7.2 kb constructs, respectively. Nuclear runon assay showed a 4-fold increase in transcription rate in response to CM. These data indicate that the human iNOS gene is transcriptionally induced by cytokines and localizes the cytokine-responsive promoter elements between 4.7 and 7.2 kb upstream in the gene. Computer analysis of the DNA sequence reveals several potential binding sites for the NF-κB transcription factor. Addition of PDTC (a NF-κB inhibitor) abrogated the CM-induced promoter activity, and gel shift assays showed inducible NF-κB DNA binding activity (data not shown), further demonstrating a role for NF-κB in regulating transcription of the human iNOS gene. Site-directed mutagenesis of 4 novel NF-κB sites from -5.2 to -6.0 kb upstream in the gene abolished the CM-induced iNOS promoter activity, thus identifying a previously unrecognized NF-κB enhancer region. Characterization of the human iNOS gene promoter is likely to provide insight into the mechanisms of cytokine signalling and suggests new strategies for modulating iNOS expression during infection or solid organ transplant rejection. Figure

Insulin increases fatty acid synthase gene transcription in human adipocytes.

The purpose of this study was to investigate the molecular mechanism whereby insulin increases expression of a key de novo lipogenic gene, fatty acid synthase (FAS), in cultured human adipocytes and hepatoma cells. RNA isolated from cultured adipocytes or from Hep G2 cells treated with or without insulin (20 nM) was analyzed. In addition, run-on transcription assays and measurements of RNA half-life were performed to determine the controlled step in FAS gene regulation by insulin. We demonstrated that FAS mRNA was expressed in both Hep G2 cells and human adipocytes. Insulin induced an approximately five- and three-fold increase in FAS mRNA content in adipocytes and hepatoma cells, respectively. Similar regulation of FAS was observed in adipocytes from lean and obese human subjects. Furthermore, we demonstrated that the induction of human FAS expression by insulin was due to increased transcription rate of the FAS gene in human adipocytes, whereas mRNA stabilization accounted for increased FAS mRNA content in hepatoma cells. In conclusion, we report here for the first time expression of human FAS mRNA and its specific transcriptional induction by insulin in cultured human adipocytes.

Posttranscriptional Regulation ofMRP/GS-XPump and γ-Glutamylcysteine Synthetase Expression by 1-(4-Amino-2-methyl-5-pyrimidinyl) methyl-3- (2-chloroethyl)-3-nitrosourea and by Cycloheximide in Human Glioma Cells

Treatment of human glioma A172 cells with 1-(4amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethy-3-nitrosourea (ACNU) for 2 to 4 hr resulted in a 2- to 3-fold increase in steady-state levels of multidrug resistance-associated protein (MRP) and γ-glutamylcysteine synthetase (γ-GCS) mRNA. Nuclear run-on assays revealed a less than 0.5-fold increase in transcription rates of these genes under the same treatment conditions, suggesting that posttranscriptional regulation plays an important role for the increased mRNA levels. In the absence of ACNU, rates ofMRPandγ-GCSmRNA degradation were similar in A172 cells as determined by incubating cells with the RNase inhibitor, Actinomycin D. ACNU treatments resulted in increasedMRPmRNA stability. Induction ofMRPandγ-GCSmRNA by ACNU apparently did not requirede novoprotein synthesis as determined by the use of protein synthesis inhibitor cycloheximide (CHX). However, CHX alone could induce accumulation ofγ-GCSmRNA, also by posttranscriptional mechanism. Taken together, these results demonstrate that (i) posttranscriptional regulation is primarily involved in the induction ofMRPandγ-GCSexpression by ACNU and CHX in human glioma cells; and (ii) despite the fact that these two genes have been reported to be frequently co-expressed, their responses to the treatments of RNA and protein synthesis inhibitors are not the same.

Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol.

Caveolae form the terminus for a major pathway of intracellular free cholesterol (FC) transport. Caveolin mRNA levels in confluent human skin fibroblasts were up-regulated following increased uptake of low density lipoprotein (LDL) FC. The increase induced by FC was not associated with detectable change in mRNA stability, indicating that caveolin mRNA levels were mediated at the level of gene transcription. A total of 924 bp of 5' flanking region of the caveolin gene were cloned and sequenced. The promoter sequence included three G+C-rich potential sterol regulatory elements (SREs), a CAAT sequence and a Sp1 consensus sequence. Deletional mutagenesis of individual SRE-like sequences indicated that of these two (at -646 and -395 bp) were essential for the increased transcription rates mediated by LDL-FC, whereas the third was inconsequential. Gel shift analysis of protein binding from nuclear extracts to these caveolin promoter DNA sequences, together with DNase I footprinting, confirmed nucleoprotein binding to the SRE-like elements as part of the transcriptional response to LDL-FC. A supershift obtained with antibody to SRE-binding protein 1 (SPEBP-1) indicated that this protein binds at -395 bp. There was no reaction at -395 bp with anti-Sp1 antibody nor with either antibody at -646 bp. The cysteine protease inhibitor N-acetyl-leu-leu-norleucinal (ALLN), which inhibits SREBP catabolism, superinhibited caveolin mRNA levels regardless of LDL-FC. This finding suggests that SREBP inhibits caveolin gene transcription in contrast to its stimulating effect on other promoters. The findings of this study are consistent with the postulated role for caveolin as a regulator of cellular FC homeostasis in quiescent peripheral cells, and the coordinate regulation by SREBP of FC influx and efflux.

Transcriptional Control of the Forkhead Thyroid Transcription Factor TTF-2 by Thyrotropin, Insulin, and Insulin-like Growth Factor I

The hormonal regulation of both thyroglobulin and thyroperoxidase promoter activity in FRTL-5 thyroid cells takes place, at least in part, through a hormone-responsive element to which the thyroid transcription factor TTF-2 binds. The TTF-2 cDNA, encoded by the titf2 locus, has recently been cloned and classified as a member of the forkhead transcription factor family. Here, we demonstrate that TTF-2 mRNA levels become undetectable in FRTL-5 thyroid cells cultured for 4 days in 0.2% serum and in the absent of thyrotropin (TSH) and insulin. Addition of TSH, insulin or insulin-like growth factor I (IGF-I) to the culture medium increases the levels of this transcription factor in a dose- and time- dependent manner and requires ongoing protein synthesis. The TSH effect is greater than that produced by insulin or IGF-I and is similar to the effect produced by the cAMP analog forskolin. The TSH and insulin effects are additive. In all cases, the mRNA levels increase is accompanied by an increase in transcription rate, as demonstrated by run-off assays. These data demonstrate that the TTF-2 mRNA is under tight hormonal control. This is consistent with an important role for TTF-2 as a mediator of the transcriptional activation of thyroid-specific genes (thyroglobulin and thyroperoxidase) by TSH via cAMP and by insulin through the IGF-I receptor.

Co-ordinated regulation of the plasma membrane calcium pump and the sarco(endo)plasmic reticular calcium pump gene expression by Ca 2+

The plasma membrane calcium pump (PMCA) and sarco(endo)plasmic reticular calcium pump (SERCA) are important components of the Ca 2+ homeostasis system responsible for intracellular Ca 2+ signaling, yet the factors which govern their expression remain unclear. Recently, we have found that overexpression of PMCA by a gene transfection approach caused a down-regulation of the SERCA pump [Liu B.F., Xu X., Fridman R., Muallem S., Kuo T.H. Consequences of functional expression of the plasma membrane calcium pump isoform 1a. J Biol Chem 1996; 271: 5536–5544]. The results suggest an interdependence between PMCA and SERCA gene expression which has prompted us to investigate further on the mechanisms that regulate the expression of these Ca 2+ pump genes in various cultured cell lines. In the present study, we have analyzed the isoforms of the PMCA and SERCA in different cells and presented evidence in favor of a co-induction of the PMCA and SERCA gene expression by second messengers, such as protein kinase C, cAMP, and Ca 2+. Ectopic expression of PMCA or SERCA led to downregulation of the endogenous forms of both pumps. Changes in the level of mRNAs were paralleled by corresponding altered pump protein contents. The Ca 2+-mediated increase of gene expression is accompanied by increased transcription rates as indicated by nuclear run-on assay. The co-ordinated induction of the PMCA and SERCA gene expression by thapsigargin was not blocked by the cytosolic application of the Ca 2+ chelator BAPTA. We conclude that genes encoding components of the major Ca 2+, transport pathways, including pumps and IP 3 receptor channels, are regulatorally linked and this link is provided by the Ca 2+ load of the ER store. This study points to the importance of gene expression as an integral component in the regulation of cellular Ca 2+ homeostasis.

Analysis of expression and promoter function of the human inducible nitric oxide synthase gene in DLD-1 cells and monkey hepatocytes

Upon cytokine induction, inducible nitric oxide synthase (iNOS) mRNA and enzyme activity in DLD-1 cells reached maximal levels at 6 and 8 h, respectively. A 3.7 kb 5′-flanking region of the human iNOS gene was used to prepare luciferase reporter constructs. Upon transfection of these constructs into DLD-1 cells and primary monkey hepatocytes, significant promoter activity was detected in the absence of cytokines, and this activity decreased with successive truncations of the human iNOS promoter. No increase in luciferase activity was observed after cytokine treatment, in spite of the fact that nuclear run-on analysis indicated that iNOS induction in DLD-1 cells was due, in part, to an increase in transcription rate. These results suggest that 3.7 kb of 5′-flanking DNA do not contain all of the elements required for transcriptional induction of the human iNOS gene. This differs from the mouse iNOS gene for which 1.7 kb of 5′-flanking DNA contain most or all of the elements that control iNOS expression in mouse macrophages. Thus, important cell- and species-specific mechanisms may exist for the control of iNOS expression.