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PubMed2002

Effect of copy number and mRNA processing and stabilization on transcript and protein levels from an engineered dual-gene operon.

To study the effect of mRNA stability and DNA copy number on protein production from a dual-gene operon, a synthetic operon containing the reporter genes gfp and lacZ under the control of the araBAD promoter was placed in pMB1-based (approximately 100 copies/cell) and F plasmid-based (approximately 1 copy/cell) vectors. DNA cassettes encoding secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the two genes. Although the copy number of the pMB1-based vectors was approximately 100-fold greater than the copy number of the F plasmid-based vectors, transcript and protein levels from the pMB1-based vector were not 100-fold greater than from the F plasmid-based vectors. In identical plasmid backbones, different combinations of mRNA control elements were used to alter steady-state levels of transcripts. Control elements that amplified the stability of one coding region relative to another amplified the ratio of protein produced from those transcripts. The effects of mRNA stability control elements were greater at low inducer concentrations, where mRNA levels limit protein production, than at high inducer concentrations. Although we can alter mRNA and protein levels through copy number, induction level, and mRNA stability control elements, some aspect of gene expression remains dependent on inherent characteristics of the coding region.

Arabinose↗
PubMed1995

Zinc inhibits turnover of labile mRNAs in intact cells.

For immediate early genes such as the c-fos proto-oncogene, mRNA breakdown is very rapid and is largely responsible for the transient nature of mRNA accumulation after transcription is stimulated. We found that in several types of cultured cells and in mice, Zn++ caused marked accumulation of c-fos mRNA and that of another labile mRNA, that encoding the tristetraprolin (TTP) protein. Exposure of TK-L cells to 100 microM ZnSO4 caused an increase of c-fos and TTP mRNA levels within 1 h that reached peak levels in 4-8 h and remained constant to 12 h. Increases in fos protein accumulation were also noted. When the cells were exposed to Zn++ for 4 h and then exposed to actinomycin D, both c-fos and TTP mRNA levels remained constant for up to 10 h, indicating that Zn++ was preventing the breakdown of both c-fos and TTP mRNA. Also, 100 microM ZnSO4 inhibited protein synthesis in TK-L cells, suggesting that the effect on mRNA accumulation could have been an indirect effect resulting from inhibited protein synthesis. Zn++ was unable to inhibit the breakdown of TTP and c-fos mRNA in vitro; however, extracts from cells exposed to Zn++ were less able to cause the breakdown of TTP and c-fos mRNAs than were extracts from control cells, again suggesting that Zn++ indirectly affects mRNA stability through inhibition of protein synthesis. These studies suggest that in addition to their effects on gene transcription, Zn++ and other divalent cations may regulate gene expression by affecting mRNA stability.

Animals↗
PubMed1993

Mechanism of interferon-gamma down-regulation of the interleukin 4-induced CD23/Fc epsilon RII expression in human B cells: post-transcriptional modulation by interferon-gamma.

It has been reported that the interleukin 4 (IL-4) specific induction of cell surface CD23 (Fc epsilon RII) is down-regulated by interferon-gamma (IFN-gamma) in monocytes and B cells. However, the molecular level at which the inhibition occurs seems to vary depending on the cell types. In normal human B cells, IFN-gamma inhibits the IL-4 induced de novo synthesis of CD23 at the level of gene expression. Analysis of inhibition kinetics suggested a rapid signal transmission by IFN-gamma. Yet the inhibitory action of IFN-gamma on CD23 mRNA accumulation appeared as a secondary response requiring a new protein synthesis. Through nuclear run-on transcription and mRNA stability studies, we further demonstrate that the IL-4 induced CD23 gene expression is down-regulated by IFN-gamma mainly at post-transcriptional levels by decreasing mRNA stability.

B-Lymphocytes↗
PubMed2003

Asp1424Asn MYH9 mutation results in an unstable protein responsible for the phenotypes in May-Hegglin anomaly/Fechtner syndrome.

May-Hegglin anomaly (MHA), Fechtner syndrome (FTNS), Sebastian syndrome (SBS), and Epstein syndrome (EPS) are a group of rare, autosomal dominant disorders characterized by thrombocytopenia, giant platelets, and Döhle-like inclusion bodies, together with variable manifestations of Alport-like symptoms that include high-tone sensorineural deafness, cataracts, and nephritis. These disorders result from mutations in the MYH9 gene, which encodes for the nonmuscle myosin heavy chain A protein (also known as NMMHC-A). To date 20 different mutations have been characterized for this gene, but no clear phenotype-genotype correlation has been established, and very little is known regarding the molecular pathogenesis of this group of diseases. Here, we describe 2 new families with MHA/FTNS phenotypes that have been characterized in terms of their mutations, protein localization in megakaryocytes, protein expression, and mRNA stability. Our findings suggest that, at least for the Asp1424Asn mutation in the MYH9 gene, the phenotypes result from a highly unstable protein. No abnormalities in protein localization or mRNA stability were observed. We hypothesize that haploinsufficiency of the MYH9 results in a failure to properly reorganize the cytoskeleton in megakaryocytes as required for efficient platelet production.

Adult↗
PubMed2003

Regulation of oxytocin receptor expression in cultured human myometrial cells by fetal bovine serum and lysophospholipids.

Oxytocin receptor (OTR) expression in human myometrium increases over 150-fold from the beginning of pregnancy to the end. In the present studies, we examined potential mechanisms of OTR up-regulation, using myometrial cells in primary culture from women in late gestation. OTR ligand-binding sites and steady-state mRNA levels were down regulated by serum starvation, and up-regulated by restoration of fetal bovine serum (FBS). Transcriptional activity of the OTR gene was the same with or without FBS treatment, but FBS increased OTR mRNA half-life about 5-fold. Lysophospholipids (lysophosphatidic acid and sphingosine 1-phosphate), which are present in serum, had similar effects as FBS. Lysophospholipid receptor mRNAs of the endothelial differentiation gene (Edg) family (Edgs 1, 3, 4, and 5) were demonstrated in myometrial cells by RT-PCR. These G protein-coupled receptors have been shown to be coupled to G(i/o) and to mediate activation of phosphoinositol 3-phosphate kinase. Indeed, the effects of the lysophospholipids and FBS were completely blocked by pertussis toxin, a G(i/o) inhibitor. Likewise, inhibition of G(i/o) signaling by elevation of intracellular cAMP or inhibition of phosphoinositol 3-phosphate kinase blocked FBS effects on OTR mRNA stability. We do not presently understand the mechanisms of OTR up-regulation in human myometrium in vivo, but the present studies might lead to the description of mRNA-stabilizing factors whose activity can be quantified in tissue samples during pregnancy to elucidate the process of OTR up-regulation.

Animals↗
PubMed1994

IL-2 mRNA levels and degradation rates change with mode of stimulation and phorbol ester treatment of lymphocytes.

Transient expression of interleukin 2 (IL-2) in activated T lymphocytes may be due to transcriptional and post-transcriptional regulation. As incubation of lymphocytes with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to mitogenic stimulation results in decreased levels of IL-2 mRNA, we asked if IL-2 mRNA stability was affected. We found that in TPA-treated cells, IL-2 mRNA was degraded more rapidly than in untreated ones whether the mitogenic stimulus was Concanavalin A (Con A), Con A plus TPA, or TPA plus ionomycin. The degradation was blocked if the TPA pre-incubation included cycloheximide. In contrast, when TPA was included as a co-mitogen, i.e. added at the same time as the mitogen, the IL-2 mRNA levels and stability significantly increased. Compared to the levels found in Con A stimulated cells, TPA plus Con A increased IL-2 mRNA levels by as much as 20-fold and the half-life by 5-fold. TPA plus ionomycin increased the message levels at least 100-fold and half-life by nearly 10-fold. These effects on IL-2 mRNA were not general because IL-2 receptor mRNA stability was not changed even though it also is transiently expressed during the course of lymphocyte activation.

Animals↗
PubMed2000

Inflammation and interleukin-6 mediate reductions in the hepatic expression and transcription of the mdr1a and mdr1b Genes.

In order to elucidate the mechanisms involved in the downregulation of mdr 1 gene expression reported in experimentally-induced inflammation, we examined the effects of experimentally-induced inflammation and interleukin-(IL-) 6 on transcriptional control of the mdr1 genes in rats. RNA, nuclear extracts, and nuclear protein fractions were isolated from livers harvested from saline or turpentine-treated male Sprague-Dawley rats or from IL-6 treated or nontreated (controls) cultured rat hepatocytes. mdr gene expression and regulation was examined by RT-PCR, mRNA stability studies, nuclear run-on analysis of transcription, and gel shift analysis of promoter-transcription factor interaction. As compared to controls, significantly lower levels of mdr1a and mdr1b mRNA and significantly decreased mdr1a and mdr1b transcription rates were observed in livers isolated from the turpentine-treated rats. In vitro treatments of cultured hepatocytes with IL-6 also suppressed mdr1a and mdr1b mRNA expression and imposed similar reductions in mdr1a and mdr1b transcriptional activity. Significant effects of IL-6 on mdr1 mRNA stability were not seen. Our results indicate that reductions in mdr1 expression in experimental models of inflammation likely occurs through altered gene transcription. Furthermore, as IL-6 was found to decrease mdr1 expression and gene transcription rates in vitro, this cytokine is likely involved in the reduction of mdr1 expression that is seen in vivo during an acute inflammatory response.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

Cell and molecular regulation of endothelin-1 production during hepatic wound healing.

During hepatic wound healing, activation of key effectors of the wounding response known as stellate cells leads to a multitude of pathological processes, including increased production of endothelin-1 (ET-1). This latter process has been linked to enhanced expression of endothelin-converting enzyme-1 (ECE-1, the enzyme that converts precursor ET-1 to the mature peptide) in activated stellate cells. Herein, we demonstrate up-regulation of 56- and 62-kDa ECE-1 3'-untranslated region (UTR) mRNA binding proteins in stellate cells after liver injury and stellate cell activation. Binding of these proteins was localized to a CC-rich region in the proximal ECE-1 3' UTR base pairs (the 56-kDa protein) and to a region between 60 and 193 base pairs in the ECE-1 3' UTR mRNA (62 kDa). A functional role for the 3' UTR mRNA/protein interaction was established in a series of reporter assays. Additionally, transforming growth factor-beta1, a cytokine integral to wound healing, stimulated ET-1 production. This effect was due to ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells, which in turn was a result of de novo synthesis of the identified 56- and 62-kDa ECE-1 3' UTR mRNA binding proteins. These data indicate that liver injury and the hepatic wound healing response lead to ECE-1 mRNA stabilization in stellate cells via binding of 56- and 62-kDa proteins, which in turn are regulated by transforming growth factor-beta. The possibility that the same or similar regulatory events are present in other forms of wound healing is raised.

3' Untranslated Regions↗
PubMed2002

Exercise, diet, and skeletal muscle gene expression.

Skeletal muscle, as a consequence of its mass and great capacity for altered metabolism, has a major impact on whole-body metabolic homeostasis and is capable of remarkable adaptation in response to various physiological stimuli, including exercise and dietary intervention. Exercise-induced increases in skeletal muscle mRNA levels of a number of genes have been reported, due to transcriptional activation and/or increased mRNA stability. The cellular adaptations to exercise training appear to be due to the cumulative effects of transient increases in gene transcription after repeated exercise bouts. The relative importance of transcriptional (mRNA synthesis) and translational (mRNA stability or translational efficiency) mechanisms for the training-induced increases in skeletal muscle protein abundance remains to be fully elucidated. Dietary manipulation, and the associated alterations in nutrient availability and hormone levels, can also modify skeletal muscle gene expression, although fewer studies have been reported. A major challenge is to understand how exercise and diet exert their effects on gene and protein expression in skeletal muscle. In relation to exercise, potential stimuli include stretch and muscle tension, the pattern of motor nerve activity and the resultant calcium transients, the energy charge of the cell and substrate availability, oxygen tension and circulating hormones. These are detected by various cellular signaling mechanisms, acting on a range of downstream targets and a wide range of putative transcription factors. A key goal in the years ahead is to identify how alterations at the level of gene expression are coupled to the changes in skeletal muscle phenotype. It is clear that gene expression, although representing a specific site of regulation, is only one step in a complex cascade from the initial stimulus to the final phenotypic adaptation and integrated physiological response.

Adaptation, Physiological↗
PubMed1989

The mechanism of cAMP-induced glucocorticoid receptor expression. Correlation to cellular glucocorticoid response.

The mechanism of regulation of glucocorticoid receptor (GR) expression by cAMP was investigated in rat hepatoma cells (HTC). Incubation of HTC cells with the cAMP-inducing agent, forskolin, caused a significant increase in the levels of both [3H]dexamethasone binding capacity and GR mRNA by about 2- to 2.4-fold within 4 h. Incubation of HTC cells with the cAMP analogue, 8-bromo-cAMP, also increased the GR mRNA level to a similar degree in a concentration-dependent manner. The increase in GR mRNA did not require ongoing translation or transcription. Determination of GR mRNA stability in 8-bromo-cAMP-induced cells showed that the message had a half-life of approximately 10 h, which is about 2.5 times longer than the GR mRNA half-life in nontreated cells (t1/2 = 4 h). These results indicate that the increased steady state level of GR mRNA induced by cAMP analogue is, at least in part, caused by increased GR mRNA stability. In both forskolin-pretreated and nontreated HTC cells, dexamethasone caused an approximately 70% down-regulation of GR protein levels. However, since forskolin induced the GR level 2- to 2.4-fold, the relative amount of GR protein remaining in cells treated with both forskolin and dexamethasone was about 2- to 2.4-fold higher compared to cells treated with dexamethasone alone. This increased GR level correlated well with the increase in inducibility of two glucocorticoid regulated genes, the endogenous tyrosine aminotransferase and the stably integrated mouse mammary tumor virus. These data suggest that relatively small changes in GR levels are reflected in parallel changes in cellular response to glucocorticoid hormones. This also implicates a limiting nature of the GR protein in determining the biological response.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

Stabilization of urokinase and urokinase receptor mRNAs by HuR is linked to its cytoplasmic accumulation induced by activated mitogen-activated protein kinase-activated protein kinase 2.

The mRNAs of urokinase plasminogen activator (uPA) and its receptor, uPAR, contain instability-determining AU-rich elements (AREs) in their 3' untranslated regions. The cellular proteins binding to these RNA sequences (ARE(uPA/uPAR)) are not known. We show here that the mRNA-stabilizing factor HuR functionally interacts with these sequences. HuR stabilized an ARE(uPA)-containing RNA substrate in vitro and stabilized in HeLa Tet-off cells both endogenous uPA and uPAR mRNAs and a beta-globin reporter mRNA containing the ARE(uPA). RNAi-mediated depletion of HuR in BT-549 and MDA-MB-231 cells significantly reduced the steady-state levels of endogenous uPA and uPAR mRNAs. Furthermore, we show that a constitutively active form of mitogen-activated protein kinase-activated protein kinase 2 (MK2), MK2-EE, has an ARE-mRNA-stabilizing effect that correlates with its ability to enhance the cytoplasmic accumulation of endogenous HuR, but not in cells cotransfected with a dominant negative version of MK2, MK2-K76R. These effects were mimicked by hydrogen peroxide treatment (oxidative stress), which resulted in the phosphorylation of endogenous MK2. In addition, hydrogen peroxide treatment enhanced the cytoplasmic binding of HuR to the ARE(uPA), which was abrogated in cells transfected with MK2-K76R. These results indicate a role for HuR and MK2 in regulating the expression of uPA and uPAR genes at the posttranscriptional level.

Antigens, Surface↗
PubMed2002

Factor VIIa induces tissue factor-dependent up-regulation of interleukin-8 in a human keratinocyte line.

Tissue factor (TF), a transmembrane receptor for the serine protease coagulation factor VII(a) (FVIIa), is the main initiator of the coagulation cascade. Through incompletely elucidated mechanisms, TF serves additional functions in tumor-associated angiogenesis and metastasis. We have studied interleukin-8 (IL-8) as a possible link between TF-FVIIa complex formation and subsequent processes. Recombinant human FVIIa induced the up-regulation of both IL-8 mRNA and protein in a FVIIa dose- and time-dependent fashion. A neutralizing antibody to TF reduced this induction by 93 +/- 5%. Active site-inhibited FVIIa had no stimulatory effect and completely blocked that of FVIIa. This confirms that the increased IL-8 production was dependent on the formation of TF-FVIIa complexes and the proteolytic activity of FVIIa. The IL-8 promoter contains DNA binding sites for nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). In response to FVIIa, the DNA binding activity of both NF-kappaB and AP-1 was enhanced in an electrophoretic mobility shift assay. In addition, the IL-8 promoter was transcriptionally activated both in a luciferase reporter system and a nuclear run-off assay. Moreover, IL-8 mRNA stability was significantly enhanced by FVIIa-induced activation of the mitogen-activated protein kinases ERK1/2 and p38. Taken together, TF-FVIIa signaling induced increased transcription as well as mRNA stabilization leading to the significant up-regulation of IL-8 protein synthesis.

Cell Line↗
PubMed2002

Cyclooxygenase-2 expression in cultured cortical thick ascending limb of Henle increases in response to decreased extracellular ionic content by both transcriptional and post-transcriptional mechanisms. Role of p38-mediated pathways.

We showed previously that decreased extracellular salt or chloride up-regulates the cortical thick ascending limb of Henle (cTALH) COX-2 expression via a p38-dependent pathway. The present studies determined that low salt medium increased COX-2 mRNA expression 3.9-fold control by 6 h in cultured cTALH, which was blocked by actinomycin D pretreatment, suggesting transcriptional regulation. Luciferase activity (normalized to beta-galactosidase activity) of the full-length (-3400) COX-2 promoter in cTALH increased from 1.8 +/- 0.3 in control media to 5.8 +/- 0.7 in low salt (n = 9; p < 0.01). Low chloride medium had similar effects as low salt has on COX-2 promoter activity. Deletion constructs -815, -512, and -410 were similarly stimulated, but -385 could not be stimulated significantly by low salt (1.8 +/- 0.3 versus 2.4 +/- 0.5, n = 10). This suggested involvement of an NF-kappaB cis-element located in this region, which was confirmed by utilizing a construct with a point mutation of this NF-kappaB-binding site that was not stimulated by low salt medium. Co-incubation of the specific p38 inhibitor, SB203580 or PD169316, inhibited a low salt-induced increase in luciferase activity of the intact COX-2 promoter (5.8 +/- 0.7 versus 1.1 +/- 0.2, n = 8 and 1.4 +/- 0.4, n = 4 respectively, p < 0.01). Mobility shift assays indicated that the low salt medium stimulated NF-kappaB binding activity, and this stimulation was inhibited by p38 inhibitors. To test whether p38 also increased COX-2 expression by increasing mRNA stability, cTALH were incubated in low salt for 2 h, and actinomycin was then added with or without SB203580. p38 inhibition led to a decreased half-life of COX-2 mRNA (from 68 to 18 min, n = 4-7, p < 0.05). Therefore, these studies indicate that p38 stimulates COX-2 expression in cTALH and macula densa by transcriptional regulation predominantly via a NF-kappaB-dependent pathway and by post-transcriptional increases in mRNA stability.

Animals↗
PubMed1990

Interferon-mediated transcriptional and post-transcriptional modulation of complement gene expression in human monocytes.

The addition of lymphoblastoid interferon alpha, fibroblast interferon beta and recombinant interferon gamma to in vitro monocyte cultures produced dose-dependent increases in transcription rates of the genes encoding the second component of complement (C2), factor B (B) and C1 inhibitor, and the abundance of their respective mRNA. Interferon gamma was the most effective at stimulating transcription of the C1-inhibitor gene whereas interferons alpha and beta were more effective at increasing the transcription of the C2 and B genes. Transcription of the C3 gene was reduced by interferon gamma. None of these cytokines altered the level of transcription of the actin gene. Interferon-induced changes in the levels of transcription of the C2, B and C1-inhibitor genes occurred rapidly, with significant changes occurring within 30 min of exposure to these cytokines. Within 4 h of removal of the interferons from the culture fluid, the level of transcription of the C1-inhibitor, C2, B and C3 genes returned to control values, as did abundance of C2, B and C3 mRNA. However, the abundance of C1-inhibitor mRNA remained elevated in interferon-gamma-treated monocytes. Combinations of interferons produced less than additive effects on the stimulation of the transcription of C2, B and C1-inhibitor genes, whereas measurements of C1-inhibitor mRNA and B mRNA showed that interferon gamma acted synergistically with interferon gamma to increase the abundance of the mRNA. Their effects on C2 mRNA abundance were less than additive. The half-lives of C1-inhibitor, C2, B and C3 mRNA were not altered by interferon alpha, whereas interferon gamma shortened the half-life of C2 mRNA by approximately 50%, and prolonged the half-lives of B and C1-inhibitor mRNA approximately twofold and fivefold, respectively. The half-life of C3 mRNA was unaltered by either interferon. These results show that the large increase in C1-inhibitor synthesis which occurs in interferon-gamma-treated monocytes, is due to a combination of increased transcription and increased C1-inhibitor mRNA stability. They also suggest that the synergistic effects of interferon alpha together with interferon gamma on C1-inhibitor and factor B synthesis is also dependent upon increased transcription and increased mRNA stability.

Complement C1 Inactivator Proteins↗
PubMed2000

RNase II removes the oligo(A) tails that destabilize the rpsO mRNA of Escherichia coli.

Polyadenylation controls mRNA stability in procaryotes, eucaryotes, and organelles. In bacteria, oligo(A) tails synthesized by poly(A) polymerase I are the targets of the 3'-to-5' exoribonucleases: polynucleotide phosphorylase and RNase II. Here we show that RNase II very efficiently removes the oligo(A) tails that can be used as binding sites by PNPase to start degradation of the rpsO mRNA. Both enzymes are impeded by the secondary structure of the transcription terminator at the 3' end of the mRNA. RNase II mostly generates tailless transcripts harboring 2 unpaired nt downstream of the transcription terminator hairpin, whereas PNPase releases molecules that exhibit a single-stranded stretch of 5-7 nt terminated by a tail of 3-5 As. The rpsO mRNAs whose oligo(A) tails have been removed by RNase II are more stable than oligoadenylated molecules that occur in strains deficient for RNase II. Moreover, the rpsO mRNA is stabilized when RNase II is overproduced. This modulation of mRNA stability by RNase II is only observed when poly(A) polymerase I is active. These in vivo data demonstrate that RNase II protects mRNAs ending by stable terminal hairpins, such as primary transcripts, from degradation by poly(A)-dependent ribonucleases.

Blotting, Northern↗
PubMed2006

Expression of the ELAV-like protein HuR in human colon cancer: association with tumor stage and cyclooxygenase-2.

There is growing body of evidence that post-translational events contribute-in addition to genetic changes-to the progression of malignant tumors. These post-translational alterations may provide targets for new therapeutic approaches. The ELAV-like protein HuR stabilizes a group of cellular mRNAs which contain AU-rich elements in their 3' untranslated region. To investigate a possible contribution of post-translational changes to the progression of colon cancer and to overexpression of COX-2, we studied expression of HuR and COX-2 a cohort of colorectal adenocarcinomas and in colon cancer cell lines. All cell lines showed an expression of HuR mRNA and protein. In tumor tissue of colon carcinomas we observed two different staining patterns of HuR: A nuclear expression in 98% as well as an additional cytoplasmic expression in 53% of cases. COX-2 was expressed in 63% of carcinomas. Cytoplasmic expression of HuR was significantly associated with increased COX-2 expression as well as with high tumor stage. In univariate Kaplan-Meier analysis, grading, tumor stage and nodal status but not HuR or COX-2 expression were prognostic factors for overall survival. Our results suggest that the overexpression of HuR in colon cancer may be part of a regulatory pathway that controls the mRNA stability of cyclooxygenase-2 and provides an interesting example for a contribution of a dysregulation of mRNA stability to the progression of colorectal cancer. Based on our results, further studies are necessary to investigate whether HuR might be a potential target for a molecular tumor therapy.

Adenocarcinoma, Mucinous↗
PubMed2007

Regulation of matrix metalloproteinase gene expression.

The metalloproteinases degrade extracellular matrix (ECM) components and activate growth factors, thereby contributing to physiological events (tissue remodeling in pregnancy, wound healing, angiogenesis) and pathological conditions (cancer, arthritis, periodontitis). The intent of this review is to bring together various studies on transcriptional and post-transcriptional control of metalloproteinase expression. Certainly, much information is known as to the cis-elements and corresponding trans-activators regulating expression of these genes. We discuss the fact that a number of the metalloproteinase promoters share common structural features and, therefore, not surprisingly are co-regulated in their expression to some extent. More recently, much effort has been devoted to understanding the role of chromatin in regulating gene expression. While this area has been understudied with respect to matrix metalloproteinase (MMP) regulation, the literature indicates a convincing role for both histone modifications and chromatin-remodeling motors in controlling expression of multiple metalloproteinases. In addition to transcriptional control, mRNA stability and protein translation also contribute to the metalloproteinase product amount. We discuss such studies and how various biological cues, including TGF-beta, regulate the levels of certain collagenases either solely through mRNA stabilization or by jointly targeting transcriptional and post-transcriptional mechanisms. We also discuss the current deficits in our knowledge, concerning tissue-specific expression and why despite elevated amounts/activity of trans-activators targeting MMP promoters in tumor cells, nevertheless, MMP expression is largely restricted to the stromal compartment. Finally, we argue for potential technologies to regulate MMP expression of utility in pathological conditions where these enzymes are aberrantly expressed.

Animals↗
PubMed1996

Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines.

IL-4 and IL-10 inhibit the cytokine production and mRNA expression by monocytes/macrophages. To investigate the molecular mechanism of the inhibitory effect on transcriptional or post-transcriptional regulation of IL-6 gene expression by IL-4 and IL-10, we studied IL-6 production, expression level of IL-6 mRNA, IL-6 promoter activity, transcriptional activity of NF-kappaB and NF-IL-6, and IL-6 mRNA stability in human monocytic cell lines, THP-1 and U937, stimulated by PMA and LPS in the absence or the presence of IL-4 or IL-10. Both IL-4 and IL-10 were seen to inhibit IL-6 production and the expression of IL-6 mRNA in both monocytic cell lines studied. In chloramphenicol acetyltransferase assays, utilizing the transient transfection of a chloramphenicol acetyltransferase reporter plasmid containing the IL-6 gene promoter, IL-4, but not IL-10, suppressed the transcriptional activity of the IL-6 gene promoter stimulated by PMA and LPS. Electrophoretic mobility shift assays showed that IL-4, but not IL-10, inhibited nuclear NF-kappaB activity, and that IL-4 and IL-10 did not affect NF-IL-6 activity. On the other hand, IL-10 enhanced the degradation of IL-6 mRNA in a mRNA stability assay. These results suggest that IL-4 may inhibit the transcription of the IL-6 gene by affecting NF-kappaB binding activity, while IL-10 may inhibit the IL-6 mRNA levels post-transcriptionally, without suppressing promoter activity. Therefore, we conclude that IL-4 and IL-10 inhibit IL-6 production by different mechanisms in human monocytic cell lines.

Base Sequence↗