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PubMed2001

Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs.

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine-Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5'-untranslated region (5'-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5'-UTR and DB region promotes accumulation of NPTII up to approximately 16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield ( approximately 23% of TSP) was obtained from a construct in which the T7g10 5'-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.

Bacteriophage T7↗
PubMed2000

Redox-regulated RNA helicase expression.

In photosynthetic organisms it is becoming increasingly evident that light-driven shifts in redox potential act as a sensor that initiates alterations in gene expression at both the level of transcription and translation. This report provides evidence that the expression of a cyanobacterial RNA helicase gene, crhR, is controlled at the level of transcription and mRNA stability by a complex series of interacting mechanisms that are redox regulated. Transcript accumulation correlates with reduction of the electron transport chain between Q(A) in photosystem II and Q(O) in cyt b(6)f, when Synechocystis sp. strain PCC 6803 is cultured photoautotrophically or photomixotrophically and subjected to darkness and/or electron transport inhibitors or illumination that preferentially excites photosystem II. crhR mRNA stability is also regulated by a redox responsive mechanism, which differs from that affecting accumulation and does not involve signaling initiated by photoreceptors. The data are most consistent with plastoquinol/cyt b(6)f interaction as the sensor initiating a signal transduction cascade resulting in accumulation of the crhR transcript. Functionally, CrhR RNA unwinding could act as a linker between redox regulated transcription and translation. The potential for translational regulation of redox-induced gene expression through RNA helicase-catalyzed modulation of RNA secondary structure is discussed.

Cyanobacteria↗
PubMed2004

17beta-estradiol enhances the production of granulocyte-macrophage colony-stimulating factor in human keratinocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is effective for impaired wound repair. Estrogen is known to enhance wound repair. We examined if 17beta-estradiol (E2) may in vitro enhance GM-CSF production in human keratinocytes. E2 and membrane-impermeable bovine serum albumin-conjugated E2 increased GM-CSF secretion, mRNA stability, and promoter activity. The element homologous to activator protein-1 (AP-1) on the promoter was responsible for the activation. E2 enhanced transcriptional activity and DNA binding of AP-1. E2 transiently generated c-Fos protein, and shifted AP-1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers in keratinocytes. E2-induced enhancement of GM-CSF secretion, mRNA stability, and promoter activity were not suppressed by estrogen receptor antagonist ICI 182,780, however, suppressed by conventional protein kinase C inhibitor Gö6976 and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK). Gö6976 and PD98059 suppressed E2-induced c-Fos expression and enhancement of DNA-binding and transcriptional activity at AP-1. E2 induced membrane translocation of protein kinase Calpha, which was suppressed by phosphatidylinositol (PI)-specific phospholipase C (PLC) inhibitor U73122. E2 stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), which was suppressed by PD98059, Gö6976, and U73122. E2 transiently generated inositol 1,4,5-triphosphate in keratinocytes, which was suppressed by U73122 and guanine nucleotide-binding protein inhibitor. These results suggest that E2 may enhance GM-CSF production via guanine nucleotide-binding protein-coupled membrane receptors and signaling cascade of PI-specific PLC/protein kinase Calpha/MEK/ERK.

Cells, Cultured↗
PubMed2006

Estrogen up-regulates neuropeptide Y Y1 receptor expression in a human breast cancer cell line.

Normal breast tissue mainly expresses the neuropeptide Y (NPY) Y2 receptor whereas primary human breast carcinomas express the Y1 receptor (Y1R) subtype. We hypothesized that activation of estrogen signaling systems plays a role in the induction of Y1R. To investigate this possibility, we used estrogen receptor-positive (ER+) human breast carcinoma cell line, MCF-7, and examined the effect of estrogen on Y1R gene expression and its signaling pathways. Saturation binding studies revealed that MCF-7 cells express high-affinity NPY receptor. NPY inhibited forskolin-stimulated adenosine 3'5'-cyclic monophosphate (cAMP) accumulation and mobilized intracellular Ca(2+) in MCF-7 cells. Chronic estrogen treatment enhanced NPY-mediated inhibition of cAMP accumulation by 4-fold and caused a significant increase in Y1R mRNA expression through ERalpha. Similarly, estrogen increased Y1R mRNA expression in T-47D (ER+) but not in MDA-MB231 or MDA-MB468 (ER-) cell lines. Cycloheximide decreased basal Y1R mRNA expression; however, it did not affect its increase by estrogen. Moreover, estrogen treatment of MCF-7 cells did not increase Y1R mRNA stability. The up-regulation of Y1R expression by estrogen is prevented by hydroxyurea but not by nocodazole or IB-MECA (cell cycle inhibitors). Lastly, NPY inhibited estrogen-induced cell proliferation through Y1R. In conclusion, MCF-7 cells express a functional Y1R coupled to both Ca(2+) and cAMP pathways. Estrogen up-regulates Y1R expression through ERalpha. This effect is independent of increased Y1R mRNA stability or new protein synthesis, and likely occurs during S phase completion of the cell cycle. Estrogen plays an important role in the up-regulation of Y1R, which in turn regulates estrogen-induced cell proliferation in breast cancer cells.

Binding, Competitive↗
PubMed1991

Perinatal rat lung catalase gene expression: influence of corticosteroid and hyperoxia.

Dexamethasone accelerates the late gestational rise in rat lung catalase activity; neonatal hyperoxia elevates rat lung catalase activity. We studied the regulation of catalase gene expression in these instances. Catalase mRNA/mg DNA increased to gestation day 22 and then fell to the concentration in adult lungs. The rate of transcription of catalase mRNA was higher on gestation day 22 than gestation day 19, whereas the half-life of catalase mRNA (approximately 7 h) was the same on both days. Dexamethasone given 48 and 24 h before expected birth (gestation 22 days) increased catalase mRNA concentration at days 20 and 22 without a change in catalase mRNA stability. Early postnatal hyperoxia (greater than 95% O2, 72 h) elevated catalase mRNA/mg DNA and doubled its half-life without changing its rate of transcription. We conclude the normal late gestational elevation of catalase activity and the increase of activity during prenatal dexamethasone treatment are regulated at the level of gene transcription. By contrast, the elevation of catalase activity during neonatal hyperoxia is mediated posttranscriptionally by increased catalase mRNA stability.

Aging↗
PubMed2006

Functional polymorphisms in the vascular endothelial growth factor gene are associated with development of end-stage renal disease in males.

This study elucidates the genetic role of vascular endothelial growth factor (VEGF) as a predisposing factor for progression of chronic kidney disease. Single-nucleotide polymorphisms were genotyped and haplotype structures were determined in the 3' untranslated region (UTR) of VEGF gene, and the distribution of each haplotype in male patients with ESRD (n=101) and healthy male control subjects (n=189) was examined. The 936C/T and 1451C/T polymorphisms in the 3' UTR were in nearly absolute linkage disequilibrium, and haplotype analysis demonstrated that they were the primary responsible single-nucleotide polymorphisms. The distribution of the 936CC-1451CC genotype was significantly more frequent among patients with ESRD than among the age-matched healthy control subjects. In addition to case-control association study, the 936CC-1451CC genotype was also associated with significantly higher plasma VEGF levels in healthy individuals, but a significant association was found only in males, not in females. We also examined the effect of the 936C-1451C haplotype on mRNA stability. Consistent with the results of plasma VEGF levels, mRNA carrying 936C-1451C haplotype showed higher stability. The 936CC-1451CC genotype in the 3' UTR showed not only susceptibility for ESRD but also higher plasma VEGF levels and mRNA stability, indicating the contribution of VEGF to chronic kidney disease progression, especially in males.

Adult↗
PubMed1989

IL-1 beta production in cultured human monocytes is regulated at multiple levels.

Cultured human monocytes and tissue macrophages exhibit a marked decrease in LPS-stimulated IL-1 production. Preincubation of monocytes in 100 U/ml IFN-gamma or in 0.25 microgram/ml cycloheximide (Cx) leads to a partial maintenance of the ability to produce IL-1 in response to subsequent stimulation with LPS, whereas preincubation in both reagents leads to a near complete maintenance of this response. These studies were performed to determine the mechanisms of these alterations in regulation of IL-1 production in cultured monocytes. In comparison to LPS-induced fresh monocytes, cells preincubated in medium for 1 day, then stimulated for 24 h with 100 ng/ml LPS, exhibited a 50% or more decrease in steady-state IL-1 beta mRNA levels. This reduction was accompanied by a four- to fivefold decrease in relative transcriptional rate, as determined by the nuclear run-on technique. The medium-aged cells also displayed greatly decreased IL-1 beta production with deficient secretion. Monocytes preincubated in IFN-gamma for 1 day before LPS stimulation exhibited a variable maintenance in IL-1 beta production, secondary both to prolonged duration of transcription and to increased IL-1 beta mRNA stability. Monocytes were preincubated for 1 day in Cx alone or in both IFN-gamma and cycloheximide, then these substances were washed out before a subsequent 24-h culture in LPS. Cx preincubation led to increases in both the peak and duration of transcription as well as to enhanced secretion of IL-1 beta protein. Monocytes preincubated in both IFN-gamma and Cx exhibited increased IL-1 beta mRNA levels due to both enhanced transcription and mRNA stability; more IL-1 beta protein secretion also was present in these cells. In summary, IFN-gamma and Cx may maintain LPS-induced IL-1 beta production in cultured human monocytes through multiple mechanisms. These alterations in regulation of IL-1 beta production during monocyte differentiation may be relevant to tissue macrophages where impaired IL-1 production may be present.

Blood Proteins↗
PubMed1994

Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae.

The polyadenylate tail of eukaryotic mRNAs is thought to influence various metabolic phenomena including mRNA stability, translation initiation, and nucleo-cytoplasmic transport. We have analyzed the fate of mRNAs following inactivation of poly(A) polymerase in Saccharomyces cerevisiae containing a temperature-sensitive, lethal mutation (pap1-1) in the gene for poly(A) polymerase (PAP1). Inactivation of poly(A) polymerase (Pap1) by shifting cells to the nonpermissive temperature resulted in the loss of at least 80% of measurable poly(A) within 60 min. Northern blot analysis revealed the disappearance of some mRNAs (CYH2 and HIS4) consistent with a role for poly(A) tails in mRNA stability. However, other mRNAs (TCM1, PAB1, ACT1, and HTB2) accumulate as poly(A)-deficient (A < approximately 25) transcripts as defined by an inability to bind oligo(dT)-cellulose. Sucrose density gradient analysis of polyribosomes revealed a twofold reduction in the amount of each size class of polyribosomes in shifted cells and a commensurate increase in free ribosomes. However, poly(A)-deficient mRNAs in shifted cells remain associated with the same size polyribosomes as poly(A)+ mRNAs in unshifted cells, indicating normal initiation of translation. RNase mapping of transcripts from pap1-1 cells revealed PAB1 mRNA to be poly(A)- whereas TCM1 exists as equal amounts of poly(A)- and poly(A)+ mRNA 60 min after shift. Interestingly, both of these classes of TCM1 mRNA appear in similar amounts in each polyribosome fraction indicating that ribosomes may not distinguish between them. These findings suggest that under conditions of excess translational capacity, poly(A)- and poly(A)+ mRNAs may initiate translation with comparable efficiencies.

Base Sequence↗
PubMed1997

Role of Escherichia coli cspA promoter sequences and adaptation of translational apparatus in the cold shock response.

A shift in growth temperature from 37 degrees C to 15 degrees C leads to a dramatic increase in the level of CspA, the major cold shock protein of Escherichia coli. To investigate the molecular basis of this induction, we considered the relevance of transcriptional and posttranscriptional controls by analyzing the steady-state levels of transcripts and the expression of reporter genes in cells carrying a set of cspA promoter fragments of variable length fused to lacZ or cat genes. We demonstrate that: (i) the core cspA promoter (from -40 to +16) responds to cold shock and a mutation at -36 increases the relative activity of the promoter at low temperature by threefold; (ii) the sequences upstream of -40 have a positive effect on expression at 37 degrees C, but no effect on the cold shock response; (iii) by virtue of their influence on mRNA stability, the downstream sequences (from +81 to +165) reduce expression at 37 degrees C and increase the intensity of the cold shock response; (iv) mutations in the GCACATCA and CCAAT motifs, present at +1/-4 and between the -10 and -35 elements, respectively, do not affect the cold shock response of the cspA promoter; (v) following cold shock, a modification of the protein synthetic machinery takes place that allows preferential translation of cspA mRNA relative to the non-cold shock cat and lacZ mRNAs. The quantitatively modest transcriptional activation shown by the core promoter of cspA following cold shock suggests that transcriptional activation can significantly contribute to cold shock induction only when coupled to posttranscriptional controls, such as alterations in mRNA stability and the translational apparatus.

Adaptation, Physiological↗
PubMed1989

Regulation of mammary differentiation by the extracellular matrix.

In multicellular organisms cell growth and differentiation are influenced by soluble factors, cell-cell interactions and cell-extracellular matrix interactions. We have used the rat mammary gland as a model system to study the role of extracellular matrix components in the regulation of milk protein gene expression. Since mammary epithelial cells differentiate on a basement membrane in vivo, we investigated the effects of basement membrane components on the expression of the milk protein genes, alpha-casein, alpha-lactalbumin, and transferrin. We have demonstrated that a basement membrane gel, as well as its major basement membrane component, laminin, induced alpha-casein and alpha-lactalbumin expression as much as 160-fold compared to tissue culture plastic. We demonstrate that laminin affects mRNA stability as well as having an effect on protein stability and secretion. Laminin interacts with mammary epithelial cells via an 68 kD cell surface receptor which is capable of interacting with the cellular cytoskeleton. In order to provide evidence that laminin affects on mammary differentiation are mediated through this receptor via the cytoskeleton, we examined the effects of cytoskeletal disrupting agents on milk protein gene expression. We demonstrate that cytochalasin D or colchicine selectively block laminin-mediated milk protein gene expression by affecting mRNA stability. Based on these experiments, we propose a model in which laminin affects mammary gene expression through interaction with cell surface receptors which interact with the cytoskeleton resulting in stabilization of mRNAs for milk protein genes.

Animals↗
PubMed2006

Characterization of the molecular mechanisms involved in the increased insulin secretion in rats with acute liver failure.

To investigate the mechanism of hyperinsulinaemia in rats with acute liver failure induced by the administration of d-galactosamine (GalN), we focused on the role of polyprimidine tract-binding protein (PTB) in islet insulin synthesis. Recent reports indicate that PTB binds and stabilizes mRNA encoding insulin and insulin secretory granule proteins, including islet cell autoantigen 512 (ICA512), prohormone convertase 1/3 (PC1/3), and PC2. In the present study, glucose-stimulated insulin secretion was significantly increased in GalN-treated rats compared to controls. Levels of mRNA encoding insulin 1, ICA512, and PC1/3 were increased in the pancreatic islets of GalN-treated rats. This mRNA level elevation was not prevented by pretreatment with actinomycin D. When the PTB-binding site in insulin 1 mRNA was incubated with the islet cytosolic fraction, the RNA-protein complex level was increased in the cytosolic fraction obtained from GalN-treated rats compared to the level in control rats. The cytosolic fraction obtained from pancreatic islets obtained from GalN-treated rats had an increased PTB level compared to the levels obtained from the pancreatic islets of control rats. These findings suggest that, in rats with acute liver failure, cytosolic PTB binds and stabilizes mRNA encoding insulin and its secretory granule proteins.

Alanine Transaminase↗
PubMed2004

Induction of cyclooxygenase-2 in macrophages by catalase: role of NF-kappaB and PI3K signaling pathways.

Induction of COX-2 by catalase in smooth muscle cells, endothelial cells, and neuronal cells has been previously reported. However, the mechanism by which catalase up-regulates COX-2 remains poorly understood. In this study, we investigated the effect of catalase on induction of COX-2 in macrophages. The addition of catalase into Raw 264.7 macrophages induced COX-2 expression that was correlated with increased COX-2 transcription and mRNA stability. Catalase also induced activation of NF-kappaB, PI3K, ERKs, p38s, or JNKs. Catalase-induced COX-2 expression was abrogated by treatment of MG-132 (a NF-kappaB inhibitor) or LY294002 (a PI3K inhibitor), but not by treatment of PD98059 (an ERK inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor). Moreover, inhibition of PI3K by LY294002 caused partial decrease of catalase-induced COX-2 transcription and steady-state COX-2 transcript levels, but not COX-2 mRNA stability. Together, these results suggest that catalase induces the expression of COX-2 in Raw 264.7 macrophages, and the induction is related with activation of NF-kappaB transcription factor and PI3K signaling pathway.

Animals↗
PubMed2003

Gene length and proximity to neighbors affect genome-wide expression levels.

Steady-state levels of mRNA in cells theoretically depend on the rate and efficiency of transcription and posttranscriptional processing, on mRNA stability, on transcriptional interference from other genes, and on poorly defined long-range chromatin effects. Although each of these cellular processes has been studied in detail for a few genes, it is not possible to predict expression levels by simply examining gene sequences. In this report, we have used a bioinformatics approach to identify critical factors that influence expression levels. To simplify the problem, we have limited our analysis to the collection of genes expressed in all tissues, because such genes provide a unique opportunity to distinguish the role of general genomic features that constrain gene expression from the effect of tissue-specific factors. Using correlation and regression techniques, we have investigated the dependence between expression level and morphological parameters (distance to neighbors, gene, mRNA or 3'-UTR length, number of exons, etc.) that can be directly related to transcription, posttranscriptional processing, mRNA stability, or transcriptional interference. We found that, on a genome-wide scale, highly expressed genes are significantly farther from their closest neighboring genes, are smaller, contain a moderate number of exons, and produce shorter mRNAs with shorter 3'-UTRs. This confirms that transcriptional and posttranscriptional processes are highly interrelated and implies that transcriptional interference plays a role in determining steady-state levels of mRNA in cells.

Computational Biology↗
PubMed1991

Transcriptional regulation of cytokine genes in nontransformed T cells. Apparent constitutive signals in run-on assays can be caused by repeat sequences.

Cytokines are immunoregulatory proteins that are secreted by T lymphocytes and other cells upon activation. A controversy exists as to whether the induction of cytokine production is mediated at the transcriptional level by the initiation of RNA synthesis or at the post-transcriptional level by the enhancement of mRNA stability. We show that in a nontransformed T cell clone the induction of all of the cytokines that are analyzed in this report is mediated transcriptionally. We also found that a constitutive signal was seen in resting cells when the probes used in the nuclear run-on assay contained some potential cross-hybridizing sequences, for example a poly (A) tract in a cDNA probe. This observation could explain the constitutive signals seen in other studies that claim cytokine production is regulated by differential mRNA stability.

Animals↗
PubMed2005

Interleukin-13 and interleukin-4 induce vascular endothelial growth factor release from airway smooth muscle cells: role of vascular endothelial growth factor genotype.

Th2 cytokines induce the release of vascular endothelial growth factor (VEGF) from cultured human airway smooth muscle cells. The objective of this study was to examine the mechanistic basis for IL-4- and IL-13-induced VEGF release and to determine whether genetic differences are responsible for donor-to-donor variability in VEGF release. We measured VEGF mRNA expression by real-time PCR, mRNA stability using actinomycin D, and promoter activity with a VEGF-promoter luciferase reporter construct. We measured IL-4- and IL-13-induced VEGF release in cells from 21 donors by ELISA, genotyped the cells for common single nucleotide polymorphisms in the IL-4R alpha (Ile50Val, Ser478Pro, and Gln551Arg) and VEGF (-460T/C, -160C/T, -152G/A, +405C/G and +936 C/T) genes, and stratified the data by IL-4R alpha and VEGF genotype. IL-4 and IL-13 increased VEGF release and VEGF mRNA expression. IL-4 also increased mRNA stability but did not affect VEGF promoter activity. There was marked donor-to-donor variability in VEGF release from smooth muscle cells. The presence of Val50, Pro478/Arg551, or the Val50/Pro478/Arg551 IL-4R alpha haplotype had little effect on VEGF release. VEGF genotype at +405 or +936 alone had no effect on VEGF release, whereas cells bearing at least one -460C/-152A/+405G VEGF allele had lower release of VEGF in response to IL-13 or IL-4 than cells with other genotypes. Our data suggest that IL-4 and IL-13 mediate their effects on VEGF expression post-transcriptionally and indicate that polymorphisms in the VEGF, but not the IL-4R alpha, gene affect VEGF release from smooth muscle cells.

Cells, Cultured↗
PubMed1992

Early meiotic transcripts are highly unstable in Saccharomyces cerevisiae.

Meiosis in Saccharomyces cerevisiae requires the induction of a large number of genes whose mRNAs accumulate at specific times during meiotic development. This study addresses the role of mRNA stability in the regulation of meiosis-specific gene expression. Evidence is provided below demonstrating that the levels of meiotic mRNAs are exquisitely regulated by both transcriptional control and RNA turnover. The data show that (i) early meiotic transcripts are extremely unstable when expressed during either vegetative growth or sporulation, and (ii) transcriptional induction, rather than RNA turnover, is the predominant mechanism responsible for meiosis-specific transcript accumulation. When genes encoding the early meiotic mRNAs are fused to other promoters and expressed during vegetative growth, their mRNA half-lives, of under 3 min, are among the shortest known in S. cerevisiae. Since these mRNAs are only twofold more stable when expressed during sporulation, we conclude that developmental regulation of mRNA turnover can be eliminated as a major contributor to meiosis-specific mRNA accumulation. The rapid degradation of the early mRNAs at all stages of the yeast life cycle, however, suggests that a specific RNA degradation system operates to maintain very low basal levels of these transcripts during vegetative growth and after their transient transcriptional induction in meiosis. Studies to identify specific cis-acting elements required for the rapid degradation of early meiotic transcripts support this idea. A series of deletion derivatives of one early meiosis-specific gene, SPO13, indicate that its mRNA contains determinants, located within the coding region, which contribute to the high instability of this transcript. Translation is another component of the degradation mechanism since frameshift and nonsense mutations within the SPO13 mRNA stabilize the transcript.

Cloning, Molecular↗
PubMed2005

Redundancy of biological regulation as the basis of emergence of multidrug resistance.

Active efflux of xenobiotics is a major mechanism of cell adaptation to environmental stress. The ATP-dependent transmembrane transporter P-glycoprotein (Pgp) confers long-term cell survival in the presence of different toxins, including anticancer drugs (this concept is referred to as multidrug resistance, or MDR). The vital importance of this mechanism for cell survival dictates the reliability and promptness of its acquisition. To fulfill this requirement, the MDR1 gene that encodes Pgp in humans must be readily upregulated in cells that express low to null levels of MDR1 mRNA prior to stress. The MDR1 gene and a stable MDR phenotype can be induced after short-term exposure of cells to a variety of cues. This effect is implemented by activation of MDR1 transcription and mRNA stabilization. The MDR1 message abundance is regulated by mechanisms generally involved in stress response, namely activation of phospholipase C, protein kinase C and mitogen-activated protein kinase cascades, mobilization of intracellular Ca2+, and nuclear factor kappa B activation. Furthermore, the proximal MDR1 promoter sites critical for induction are not unique for the MDR1 gene; they are common regulatory elements in eukaryotic promoters. Moreover, MDR1 induction can result from activation of (an) intermediate gene(s) whose product(s), in turn, directly activate(s) the MDR1 promoter and/or cause(s) mRNA stabilization. Redundancy of signal transduction and transcriptional mechanisms is the basis for the virtually ubiquitous inducibility of the MDR1 gene. Thus, the complex network of MDR1 regulation ensures rapid emergence of pleiotropic resistance in cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Differential regulation of the expression of fast-type sarcoplasmic-reticulum Ca(2+)-ATPase by thyroid hormone and insulin-like growth factor-I in the L6 muscle cell line.

The aim of this study was to investigate the mechanism(s) underlying the thyroid-hormone (L-tri-iodothyronine, T3)-induced elevation of fast-type sarcoplasmic-reticulum Ca(2+)-ATPase (SERCA1) levels in L6 myotubes and the potentiating effect of insulin-like growth factor-I (IGF-I) [Muller, van Hardeveld, Simonides and van Rijn (1991) Biochem. J. 275, 35-40]. T3 increased the SERCA1 protein level (per microgram of DNA) by 160%. The concomitant increase in the SERCA1 mRNA level was somewhat higher (240%). IGF-I also increased SERCA1 protein (110%) and mRNA levels (50%), whereas IGF-I + T3 increased SERCA1 protein and mRNA levels by 410% and 380% respectively. These SERCA1 mRNA analyses show that the more-than-additive action of T3 and IGF-I on SERCA1 expression is, at least in part, pre-translational in nature. Further studies showed that the half-life of SERCA1 protein in L6 cells (17.5 h) was not altered by T3. In contrast, IGF-I prolonged the half-life of SERCA1 protein 1.5-1.9-fold, which may contribute to the disproportional increase in SERCA1 protein content compared with mRNA by IGF-I. Measurements of SERCA1 mRNA half-life (as determined by actinomycin D chase) showed no difference from the control values (15.5 h) in the presence of T3 or IGF-I alone. When T3 and IGF-I were both present, the SERCA1 mRNA half-life was prolonged 2-fold. No significant effects of T3 and IGF-I were observed on the half-life of total protein (37.4 h) and total RNA (37.0 h). The absence of an effect of T3 on SERCA1 protein and mRNA stability, when it was present alone, suggested transcriptional regulation, which was confirmed by nuclear run-on experiments, showing a 3-fold increase in transcription frequency of the SERCA1 gene by T3. We conclude that the synergistic stimulating effects of T3 and IGF-I on SERCA1 expression are the result of both transcriptional and post-transcriptional regulation. T3 acts primarily at the transcriptional level by increasing the transcription frequency of the SERCA1 gene, whereas IGF-I seems to act predominantly at post-transcriptional levels by enhancing SERCA1 protein and mRNA stability, the latter, however, only in the presence of T3.

Animals↗