Enhanced Reporter Gene Expression Research Articles

Enhancement of Reporter-Gene Expression by Insertions of Two Introns in Maize and Tobacco Protoplasts

The effects of two-intron insertions on reporter-gene expression were examined in transient assays in maize and tobacco. The first introns of a rice gene for phospholipase D (PLD), a maize gene for ubiquitin and a castor bean gene for catalase were tested with a gene for β-glucuronidase (GUS) or a gene for luciferase driven by a cauliflower mosaic virus 35S promoter (35S-GUS or 35S-Luc), the promoter of the maize ubiquitin (Ubi-GUS) or the promoter of a maize pyruvate, orthophosphate dikinase (PPDK-GUS). Although the enhancing effect varied, several intron combinations synergistically enhanced the reporters. When the first and second introns of the PLD gene were inserted in tandem, the reporter was synergistically enhanced. These results imply that expression enhancement by two introns may be observed in wide combinations of promoters, introns, and structural genes and that such enhancement plays certain roles in regulation of gene expression in higher plants.

PACT-mediated enhancement of reporter gene expression at the translational level.

The cellular protein, PACT, can directly activate protein kinase (PKR) in vitro by the interaction of PACT domain 3 with PKR. In contrast, in vivo, PACT-mediated PKR activation and concomitant inhibition of protein synthesis require additional cellular stresses. We observed that without such stresses, cotransfection of a PACT expression vector with various reporter genes enhances their levels of expression. This effect was promoter and inducer-independent and PACT specific and mediated by PACT domains 1 and 2. PACT did not increase the level of the reporter mRNA but enhanced its translation by suppressing phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) caused by the transfection process. To further examine the phenomenon, we generated cell lines expressing a PACT mutant containing only domains 1 and 2. Reporter gene expression was higher and eIF2alpha phosphorylation was lower in such cell lines compared with the corresponding control cells. Thus, different domains of PACT can either promote or inhibit translation by appropriately modulating the status of eIF2alpha phosphorylation.

Focused ultrasound (HIFU) induces localized enhancement of reporter gene expression in rabbit carotid artery.

The development of accurate, safe, and efficient gene delivery remains a major challenge towards the realization of gene therapeutic prevention and treatment of cardiovascular diseases. In this study, we investigated the ability of high-intensity focused ultrasound (HIFU), a form of mechanical wave transmission, to act as a noninvasive tool for the enhancement of in vivo gene transfer into rabbit carotid arteries. Segments of the common carotid arteries of New Zealand white rabbits were isolated and infused with plasmid DNA encoding the reporter beta-galactosidase either with or without the addition of ultrasound contrast agent consisting of small (approximately 2-5 microm) gas-filled human albumin microspheres to augment cavitation. Infused arteries were exposed to pulsed ultrasound for 1 min (frequency 0.85 MHz, burst length 50 ms, repetition frequency 1 Hz, duration 60 s, peak pressure amplitude of 15 MPa). At 6.3 MPa, HIFU enhanced gene expression eight-fold, and 17.5-fold in the presence of contrast. We found increasing amounts of beta-galactosidase expression in the carotid vessel with increasing pressure amplitude. This dose-response relation was present with and without contrast. Without contrast, no vessel damage was detected up to 15 MPa, while the addition of contrast induced side effects above a threshold of 6.3 MPa peak pressure. The entire procedure was feasible and safe for the animals, and the results suggest that HIFU has the potential to assist in the noninvasive spatial regulation of gene transfer into the vascular system.

Enhancements in Gene Expression by the Choice of Plasmid DNA Formulations Containing Neutral Polymeric Excipients

Formulations containing maltodextrin (2% w/v) were identified to facilitate intramuscular (im) delivery of plasmid DNA in mice using the reporter genes luciferase and chloramphenicol acetyltransferase (CAT) and the therapeutic gene of erythropoietin (EPO) as monitors of transfection efficiency. Even though considerable variability in gene expression was observed in animals, a 5–8-fold enhancement of reporter gene expression was observed with this excipient compared with saline formulations of DNA. In a therapeutically significant experiment, a single im injection of an EPO plasmid formulation containing 2% (w/v) maltodextrin resulted in a significant and prolonged elevation of the hematocrit levels of mice compared with control DNA in saline. Biophysical studies with Fourier transform infrared (FTIR) spectroscopy, isothermal titration, and differential scanning calorimetry (DSC) suggested a weak interaction between DNA and maltodextrin as well as a thermal stabilizing effect on the DNA. These in vivo and biophysical results with maltodextrin are comparable to those reported previously with other nonionic polymers, such as poly(vinyl pyrrolidone) and poloxamers, and indicate that maltodextrin is an additional nonionic excipient that displays the property of gene expression enhancement. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91: 1371–1381, 2002

Use of perfluorocarbon (fluorinert) to enhance reporter gene expression following intratracheal instillation into the lungs of Balb/c mice: Implications for nebulized delivery of plasmids

Perfluorocarbons combine high respiratory gas dissolving capabilities with extreme chemical and biological inertness and therefore offer an attractive option as an excipient in the area of pulmonary therapeutics. Perfluorocarbons have also been shown to ‘float’ mucus, because of their high densities (1.9–2.5 g/mL), which may hold potential in gene delivery for cystic fibrosis patients, in terms of enhancing penetration through highly viscous mucus and thereby providing access to target epithelial cells to correct the gene defect. Additionally, their low surface tension allows for better dispersion. A commonly available perflurocarbon, heptacosafluorotributylamine (Fluorinert), was used to deliver either plasmid DNA (pDNA) alone or cationic‐lipid‐complexed plasmid DNA to the lungs of Balb/c mice by direct intratracheal instillation. The complexes consisted of supercoiled (SC) plasmid DNA (4.7 Kb, 0.625mg/mL) and lipid (ethyldimyristoyl phosphatidylcholine [EDMPC]/cholesterol [1:1 mole ratio], with pDNA (3:1mg pDNA/mM EDMPC in 20mM Tris‐HCl pH 8.0) expressing chloramphenicol acetyl transferase (CAT) or β‐galactosidase (β‐Gal). pDNA alone was supplemented with 14% w/v Fluorinert. Cationic lipid/pDNA complexes were supplemented with 3, 8, and 14% w/v Fluorinert. Results showed that the CAT expression from pDNA alone was enhanced 24× using 14% w/v Fluorinert, whereas that from the cationic‐lipid‐formulated pDNA was enhanced 7× using 14% w/v Fluorinert. Immunohistochemistry showed that β‐Gal expression was primarily from epithelial cells and not from F4/80 or MAC3 antigen‐stained cells (predominantly macrophages), indicating efficient delivery. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1336–1344, 2001

DNA elements of the type 1 adenylyl cyclase gene locus enhance reporter gene expression in neurons and pinealocytes.

The Ca2+-stimulated type 1 adenylyl cyclase (AC1) contributes to several forms of synaptic plasticity and is the only known neurospecific adenylyl cyclase. Furthermore, the protein and mRNA levels of AC1 undergo a circadian oscillation in the pineal gland, and AC1 may play a pivotal role in regulating nocturnal melatonin synthesis. To better understand the expression of AC1, we isolated mouse genomic DNA clones of AC1. The transcription and translation start regions of mouse AC1 share extensive homologies with the bovine counterpart. The upstream proximal region has potential binding sites for transcription factors, including the steroid receptor family, the E-box factors, and Sp1. A 280-bp fragment that contains the transcription start site directed reporter gene expression in cultured cortical neurons and pinealocytes functioning as a basal neuro- and pineal-directed promoter. Interestingly, pinealocyte expression of the reporter gene was inhibited by increases in cAMP. This cAMP sensitivity may explain why AC1 mRNA in the pineal is low at night when cAMP is elevated and high during the day when cAMP signals drop. An adjacent 330-bp fragment interacted specifically with nuclear factor(s) that we designate binary E-box factor (BEF). Methylation interference and DNase I footprinting identified the BEF-binding site sequence as 5'-CCAAGGTCACGTGGC-3'. When linked to the basal tissue-directed promoter, this 15-bp sequence further enhanced reporter expression in neurons and pinealocytes. We propose that this 15-bp sequence may contribute to increased expression of AC1 in neurons and pinealocytes relative to other cells.

Enhanced reporter gene expression in the rat brain from helper virus-free HSV-1 vectors packaged in the presence of specific mutated HSV-1 proteins that affect the virion

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off promoters in HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus, recombinant gene expression remains short-term in the absence of ∼99% of the HSV-1 genome. To resolve this paradox, we hypothesized that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. This study evaluated expression from HSV-1 vectors, containing neuronal-specific promoters, that were packaged in the presence of specific mutated HSV-1 proteins that affect the virion. The mutated HSV-1 proteins that were examined included two protein kinases (U L13 and U S3), the virion host shut-off factor ( vhs), the transactivator of immediate early promoters (VP16), and a virion protein that affects RNA metabolism (U S11). Helper virus-free packaging could occur in the presence of each mutated protein alone or specific combinations of two or three mutated proteins. In BHK and PC12 cells, vectors packaged in the presence of each mutated protein increased (∼2-fold) the level of expression per cell, and vectors packaged in the presence of specific combinations of mutated proteins supported larger (4–7-fold) increases. In the rat striatum, vectors packaged in the presence of a mutated U S3 displayed enhanced gene transfer (13–18-fold increases in the number of cells at 4 days), and vectors packaged in the presence of mutated U L13 or VP16 enhanced long-term expression (2 months). Vectors packaged in the presence of mutated vhs or U S11 displayed minimal changes in expression.

Subcytocidal attack by staphylococcal alpha-toxin activates NF-kappaB and induces interleukin-8 production.

Formation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression following toxin treatment. Electrophoretic mobility shift and immunofluorescence assays demonstrated that IL-8 secretion was preceded by activation of NF-kappaB. Transfection experiments conducted with p65/p50 double-deficient cells showed that activation of the IL-8 promoter/reporter by toxin was absolutely dependent on NF-kappaB. In contrast, this transcription factor was not required for lesion repair. Attack of cells by low doses of a pore-forming toxin can lead to transcriptional gene activation, which is followed by production of mediators that may contribute to the initiation and propagation of inflammatory lesions.

In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound.

Gene therapy as a form of molecular medicine is expected to have a major impact on medical treatments in the future. However, the clinical use of gene therapy today is hampered by inadequate gene delivering systems to ensure sufficient, accurate and safe DNA uptake in the target cells in vivo. Nonviral transfection methods might have the advantage of safe application, but it would be helpful to increase their transfection rates, especially in vivo. In this study, we show that focused ultrasound provides an enhanced transfer of DNA plasmids in vitro and in vivo. In vitro, the beta-galactosidase and luciferase DNA reporter plasmid were transfected into four cell lines (NIH 3T3 fibroblasts, malignant melanoma Mewo, HeLa, Dunning prostate tumor R3327-AT1). Ultrasound induced a 55- (Mewo) to 220-fold (AT1) stimulation resulting in transfection efficiencies in vitro between 2% (Mewo) and 12% (AT1). The in vivo stimulation was assessed in the Dunning prostate tumor R3327-AT1 implanted subcutaneously in Copenhagen rats using the beta-galactosidase reporter. After intratumoral DNA injection, focused ultrasound induced a 10-fold increase of beta-galactosidase positive cells in histology and a 15-fold increase of beta-galactosidase protein expression in the ELISA assay. In contrast, ultrasound was not found to enhance reporter gene expression after intravenous plasmid application. Because ultrasound waves can be focused on different anatomical locations in the human body without significant adverse effects, the control of DNA transfer by focused ultrasound is a promising in vivo method for spatial regulation of gene-based medical treatments.

PAH effects on grass shrimp, Palaeomonetes pugio: interactions with the ecdysteroid system

The steroid hormone ecdysone controls molting and differentiation in Arthropods. We used a multi-tiered approach to investigate whether polycyclic aromatic hydrocarbons (PAHs) interact with the functional Ecdysone Receptor (EcR) in vitro, and whether PAHs have molting or reproductive effects in vivo. We focused on four PAHs: pyrene, benzo[a]pyrene, benzo[b]fluoranthene, and chrysene. A reporter gene assay using a Chinese hamster ovary cell line which stably expresses the EcR/RXR complex, showed that the four PAHs had no agonistic activity, but were able to enhance reporter gene expression when given in conjuction with an ecdysteroid agonist, muristerone A. An ecdysone-responsive Drosophila cell line (Cl 8+) showed that PAHs alone had no effect, but were able to enhance the differentiation response in the presence of muristerone A. Finally, ablated grass shrimp ( Palaeomonetes spp.) were exposed to 100 nM chrysene, and molted approximately 12 h earlier than the vehicle controls. Since chrysene was able to enhance reporter gene activation and the Cl 8+ cellular response in the presence of an ecdysteroid, the accelerated molting correlates well with the in vitro assays. A chronic 8 week exposure study on intact shrimp is currently underway to determine molting and reproductive endpoints, as well as P450 and lipovitellin levels.

Ultrasound enhances reporter gene expression after transfection of vascular cells in vitro.

Restenosis after percutaneous coronary intervention remains a serious clinical problem. Progress in local gene therapy to prevent restenosis has been hindered by concerns over the safety and efficacy of viral vectors and the limited efficiency of nonviral techniques. This study investigates the use of adjunctive ultrasound to enhance nonviral gene delivery. Cultured porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were transfected with naked or liposome-complexed luciferase reporter plasmid for 3 hours. Ultrasound exposure (USE) for 60 seconds at 1 MHz, 0.4 W/cm2, 30 minutes into this transfection period enhanced luciferase activity 48 hours later by 7.5-fold and 2. 4-fold, respectively. Luciferase activity after lipofection of ECs was similarly enhanced 3.3-fold by adjunctive USE. USE had no effect on cell viability, although it inhibited VSMC but not EC proliferation. Adjunctive USE was associated with enhanced transgene expression in VSMCs and ECs and reduced VSMC but not EC proliferation in vitro, which suggests that ultrasound-assisted local gene therapy has potential as an antirestenotic therapy.

Evidence for a functional repeat polymorphism in the promoter of the human NRAMP1 gene that correlates with autoimmune versus infectious disease susceptibility

A polymorphism in the promoter of human NRAMP1 encodes a Z-DNA forming dinucleotide repeat with four alleles: (1) t(gt)5ac(gt)5ac(gt)11g; (2) t(gt)5ac(gt)5 ac(gt)10g; (3) t(gt)5ac(gt)5ac(gt)9g; and (4) t(gt)5ac(gt)9g. Alleles 1 and...

A PEA3 Site Flanked by SP1, SP4, and GATA Sites Positively Regulates the Differentiation-Dependent Expression ofBrachyuryin Embryonal Carcinoma P19 Cells

The promoter sequence ofBrachyurywas analyzed in mouse embryonal carcinoma P19 cells. The sequence up to −267 bp relative to the transcription start site was sufficient to enhance reporter gene expression depending on the mesodermal differentiation of P19 cells. Footprint analysis by nuclear extract showed binding of a GATA protein and SP4 and mutation of their sites reduced reporter gene expression. Gel-retardation assay in the presence of a series of double-stranded DNA fragments as the competitors showed SP1 and Est sites additionally. Deletion of either sites reduced the reporter gene expression, showing that they are cooperative. Depletion of PEA3 (a transcription factor of the Est family) with a specific antibody diminished the retarded bands only for the nuclear extract from differentiated P19 cells. Thus, the PEA3 site supported by SP1, SP4, and GATA sites positively regulates the differentiation-dependent expression ofBrachyuryin P19 cells.

Myelin Proteolipid Protein Intron 1 Sequences Do Not Appear to Enhance Myelin Proteolipid Protein Gene Transcription

Sequences from the first intron of the mouse myelin proteolipid protein (PLP) gene were examined for their ability to modulate PLP gene expression. Glial (N20.1) or nonglial (NIH 3T3) cells were transiently transfected with constructs that contained 1.4 kb of PLP promoter sequence driving luciferase reporter gene expression, as well as various portions of PLP intron 1 DNA. Although these same PLP intron 1 fragments enhanced reporter gene expression from a heterologous basal promoter in a previous study, the results reported here demonstrate that they do not augment PLP promoter activity. Thus, the regulation of PLP cell-type-specific expression, conferred by the first intron, appears to be mediated by an enhancer-independent mechanism.

Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor.

Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells. We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 microgram/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes: (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency. Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of foreign genes and inhibition of specific nucleases may facilitate transfection.

GA-binding Protein-dependent Transcription Initiator Elements: EFFECT OF HELICAL SPACING BETWEEN POLYOMAVIRUS ENHANCER A FACTOR 3(PEA3)/Ets-BINDING SITES ON INITIATOR ACTIVITY

Many eukaryotic RNA polymerase II promoters contain initiator elements which direct accurate transcription in a TATA-independent manner. The PEA3/Ets-binding site (PEA3/EBS) is a common enhancer element in eukaryotic genes and is also found near the transcriptional start sites of many TATA-less promoters. We demonstrate that two PEA3/EBSs driving expression of the luciferase reporter gene, function as a minimal transcriptional initiator element. Maximal levels of transcription was achieved when two PEA3/EBSs, in either orientation, were located on the same face of the DNA helix, and the sites could be separated by up to three helical turns. In vitro transcription start sites directed by PEA3/EBS elements were clustered on either side of the upstream PEA3/EBS and were abolished by immunodepletion of GA-binding protein (GABP) from FM3A cell nuclear extracts. In vivo, co-transfection of GABPalpha and GABPbeta expression vectors enhanced reporter gene expression driven from PEA3/EBS initiator elements. Like other initiator elements, the PEA3/EBS elements were activated synergistically by upstream Sp1-binding sites. Thus, our results establish GABP as both a transcriptional activator factor and as an initiator factor.

Oxygen-regulated Transferrin Expression Is Mediated by Hypoxia-inducible Factor-1

Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1beta mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1beta-deficient subline Hepa1C4, as well as antibodies raised against HIF-1alpha and ARNT/HIF-1beta confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5' HBS was more efficient in binding HIF-1 than the 3' HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer.

Determinants of Phenylethanolamine-N-methyltransferase Expression

Axelrod and Wurtman, in 1960, demonstrated that the decline in adrenal phenylethanolarnine-N-methyltransferase (PNMT) enzymatic activity following hypophysectomy in rats could be reversed by administration of the synthetic glucocorticoid, dexamethasone. It was more than two decades later those glucocorticoids were shown to increase steady-state levels of PNMT mRNA, primarily by enhancing the rate of PNMT gene transcription. Neurally mediated regulation of PNMT expression in adrenal chromaffin cells occurs predominantly through the influence of cholinergic stimuli on nicotinic and muscarinic receptors. A single neurotransmitter, acetylcholine, is capable of acting through two separate intracellular signaling pathways to stimulate transcription of the PNMT gene. Although the importance of cell-specific determinants has been recognized in a number of systems, only recently have it has been begun to characterize those sequences responsible for the highly restricted expression of PNMT. Not surprisingly, the PNMT gene likewise possesses information that directs the site of its expression. Transient transfections of upstream PNMT promoter regions were conducted utilizing an array of pheochromocytoma, neuroblastoma, glioma, fibroblast, and myoblast continuous cell lines. Deletion of the most distal element produces enhancement of reporter gene expression in nonadrenergic, nonchromaffin cell types. The PNMT mid and proximal cell-specific regions each share approximately 70% sequence similarity with a derived consensus sequence for neural specific silencers. Deletion of the regions of the PNMT promoter between -647 and -572 bp and between -572 and -537 bp produces enhancement of reporter gene constructs transfected into nonneuronal cell types, for example, C6 glioma and L6 myoblast lines. This chapter has demonstrated that the major influences establishing the physiological level of PNMT expression can be classified as hormonal, neural, and cell-specific in nature.

Modulation of transcription of the rat fibronectin gene by cell density

The fibronectin (FN) gene is under complex regulatory control in vitro and in vivo. Sequences from the rat FN gene directed efficient expression of a lacZ reporter gene product, β-galactosidase, in NIH/3T3 mouse fibroblasts. Stable transfectants were generated to facilitate studies of gene regulation by cell growth state. The expression of FN-lacZ constructs increased approximately twofold when cultures attained confluence, relative to total protein. The magnitude of this increase correlates well with that observed for FN mRNA levels and protein synthesis rate. Fragments containing 4.9, 0.9, or 0.3 kbp upstream of the transcription start site are equally responsive to cell density and/or cell contact. Deletion of a cAMP-responsive element enhanced the response, suggesting a negative role for this sequence motif and demonstrating that the FN gene is regulated by cell density at the transcriptional level. The effect of high cell density is apparently different from decreased growth rate, as incubation with low serum did not result in increased expression of the lacZ reporter. Finally, conditioned medium from dense cells did not enhance reporter gene expression in sparse cells, suggesting that the density signal is not transmitted via a soluble factor. © 1996 Wiley-Liss, Inc.

Modulation of transcription of the rat fibronectin gene by cell density

The fibronectin (FN) gene is under complex regulatory control in vitro and in vivo. Sequences from the rat FN gene directed efficient expression of a lacZ reporter gene product, beta-galactosidase, in NIH/3T3 mouse fibroblasts. Stable transfectants were generated to facilitate studies of gene regulation by cell growth state. The expression of FN-lacZ constructs increased approximately twofold when cultures attained confluence, relative to total protein. The magnitude of this increase correlates well with that observed for FN mRNA levels and protein synthesis rate. Fragments containing 4.9, 0.9, or 0.3 kbp upstream of the transcription start site are equally responsive to cell density and/or cell contact. Deletion of a cAMP-responsive element enhanced the response, suggesting a negative role for this sequence motif and demonstrating that the FN gene is regulated by cell density at the transcriptional level. The effect of high cell density is apparently different from decreased growth rate, as incubation with low serum did not result in increased expression of the lacZ reporter. Finally, conditioned medium from dense cells did not enhance reporter gene expression in sparse cells, suggesting that the density signal is not transmitted via a soluble factor.